Anti-pd-l1 antibodies and uses thereof

ABSTRACT

The present application relates to anti-PD-L1 antibodies or antigen binding fragments thereof, nucleic acid encoding the same, therapeutic compositions thereof, and their use to enhance T-cell function to upregulate cell-mediated immune responses and for the treatment of T cell dysfunctional disorders, such as tumor immunity, for the treatment of and cancer.

FIELD OF THE INVENTION

The present application relates to anti-PD-L1 antibodies or antigenbinding fragments thereof, nucleic acid encoding the same, therapeuticcompositions thereof, and their use to enhance T-cell function toupregulate cell-mediated immune responses and for the treatment of Tcell dysfunctional disorders, such as tumor immunity, for the treatmentof and cancer.

BACKGROUND OF THE INVENTION Lymphocyte Development and Activation

The two major types of lymphocytes in humans are T (thymus-derived) andB (bone marrow derived. These cells are derived from hematopoietic stemcells in the bone marrow and fetal liver that have committed to thelymphoid development pathway.

The progeny of these stem cells follow divergent pathways to mature intoeither B or T lymphocytes. Human B-lymphocyle development takes placeentirely within the bone marrow. T cells, on the other hand, developfrom immature precursors that leave the marrow and travel through thebloodstream to the thymus, where they proliferate and differentiate intomature T lymphocytes.

Mature lymphocytes that emerge from the thymus or bone marrow are in aquiescent, or “resting” state, i.e., they are mitotically inactive. Whendispersed into the bloodstream, these “naive” or “virgin” lymphocytes,travel into various secondary or peripheral lymphoid organs, such as thespleen, lymph nodes or tonsils. Most virgin lymphocytes have aninherently short life span and die without a few days after leaving themarrow or thymus. However, if such a cell receives signals that indicatethe presence of an antigen, they may activate and undergo successiverounds of cell division. Some of the resulting progeny cells then revertto the resting state to become memory lymphocytes—B and T cells that areessentially primed for the next encounter with the stimulating allergen.The other progeny of activated virgin lymphocytes are effector cells,which survive for only a few days, but carry out specific defensiveactivities.

Lymphocyte activation refers to an ordered series of events throughwhich a resting lymphocyte passes as it is stimulated to divide andproduce progeny, some of which become effector cells. A full responseincludes both the induction of cell proliferation (mitogenesis) and theexpression of immunologic functions. Lymphocytes become activated whenspecific ligands bind to receptors on their surfaces. The ligands aredifferent for T cells and B cells, but the resulting intracellularphysiological mechanisms are similar.

Some foreign antigens themselves can induce lymphocyte activation,especially large polymeric antigens that cross-link surfaceimmunoglobulins on B-cells, or other glycoproteins T-cells. However,most antigens are not polymeric and even direct binding to B-cells inlarge numbers fail to result in activation. These more common antigensactivate B cells when they are co-stimulated with nearby activatedhelper T-lymphocytes. Such stimulation may occur from lymphokinessecreted by the T-cell, but is transmitted most efficiently by directcontact of the B cell with T-cell surface proteins that interact withcertain B-cell surface receptors to generate a secondary signal.

T-Cells

T lymphocytes do not express immunoglobulins, but, instead detect thepresence of foreign substances by way of surface proteins called T-cellreceptors (TCR). These receptors recognize antigens by either directcontact or through influencing the activity of other immune cells.Together with macrophages, T cells are the primary cell type involved inthe cell-mediated immunity.

Unlike B-cells, T-cells can detect foreign substances only in specificcontexts. In particular, T-lymphocytes will recognize a foreign proteinonly if it first cleaved into small peptides, which are then displayedon the surface of a second host cell, called an antigen-presenting cell(APC). Many types of host cells can present antigens under someconditions but certain types are more specifically adapted for thispurpose and are particularly important in controlling T-cell activity,including macrophages and other B-cells. Antigen presentation depends inpart on specific proteins, called major histocompatibility complex (MHC)proteins, on the surface of the presenting cells. Thus, to stimulatecell-mediated immunity, foreign peptides must be presented to T-cells incombination with MHC peptides, and this combination must be recognizedby a T-cell receptor.

There are two significant T-cell subsets: cytotoxic T lymphocytes (T_(c)cells or CTLs) and helper T cells (T_(H)) cells, which can roughly beidentified on the basis of cell surface expression of the marker CD8 andCD4. T_(c) cells are important in viral defense, and can kill virusesdirectly by recognizing certain cell surface expressed viral peptides.T_(H) cells promote proliferation, maturation and immunologic functionof other cell types, e.g., lymphokine secretion to control activities ofB cells, macrophages and cytotoxic T cells. Both virgin and memoryT-lymphocytes ordinarily remain in the resting state, and in this statethey do not exhibit significant helper or cytotoxic activity. Whenactivated, these cells undergo several rounds of mitotic division toproduce daughter cells. Some of these daughter cells return to theresting state as memory cells, but others become effector cells thatactively express helper or cytotoxic activity. These daughter cellsresemble their parents: CD4+ cells can only product CD4+ progeny, whileCD8+ cells yield only CD8+ progeny. Effector T-cells express cellsurface markers that are not expressed on resting T-cells, such as CD25,CD28, CD29, CD40L, transferrin receptors and class II MHC proteins. Whenthe activating stimuli is withdrawn, cytotoxic or helper activitygradually subsides over a period of several days as the effector cellseither die or revert to the resting state. Similar to B-cell activation,T-lymphocyte responses to most antigens also require two types ofsimultaneous stimuli. The first is the antigen, which if appropriatelydisplayed by MHC proteins on an antigen-presenting cell, can berecognized and bound by T-call receptors. While this antigen-MHC complexdoes send a signal to the cell interior, it is usually insufficient toresult in T-cell activation. Full activation, such as occurs with helperT-cells, requires costimulation with other specific ligands calledcostimulators that are expressed on the surface of theantigen-presenting cell. Activation of a cytotoxic cell, on the otherhand, generally requires IL-2, a cyokine secreted by activated helper Tcells.

PD-1 Pathway

An important negative co-stimulatory signal regulating T cell activationis provided by programmed death-1 receptor (PD-1) (CD279), and itsligand binding partners PD-L1 (B7-H1, CD274) and PD-L2 (B7-DC, CD273).The negative regulatory role of PD-1 was revealed by PD-1 knock outs(Pdcd1^(-/-)), which are prone to autoimmunity. Nishimura et al,Immunity JJ: 141-51 (1999); Nishimura et al, Science 291: 319-22 (2001).PD-1 is related to CD28 and CTLA-4, but lacks the membrane proximalcysteine that allows homodimerization. The cytoplasmic domain of PD-1contains an immunoreceptor tyorine-based inhibition motif (ITIM,V/IxYxxL/V). PD-1 only binds to PD-L1 and PD-L2. Freeman et al, J. Exp.Med. 192: 1-9 (2000); Dong et al, Nature Med. 5: 1365-1369 (1999);Latchman et al, Nature Immunol 2: 261-268 (2001); Tseng et al, J. Exp.Med. 193: 839-846 (2001).

PD-1 can be expressed on T cells, B cells, natural killer T cells,activated monocytes and dendritic cells (DCs). PD-1 is expressed byactivated, but not by unstimulated human CD4⁺ and CD8⁺ T cells, B cellsand myeloid cells. This stands in contrast to the more restrictedexpression of CD28 and CTLA-4. Nishimura et al, Int. Immunol. 8: 773-80(1996); Boettler et al, J. Virol. 80: 3532-40 (2006). There are at least4 variants of PD-1 that have been cloned from activated human T cells,including transcripts lacking (i) exon (ii) exon 3, (iii) exons 2 and 3or (iv) exons 2 through 4. Nielsen et al, Cell. Immunol. 235: 109-16(2005). With the exception of PD-IΔex3, all variants are expressed atsimilar levels as full length PD-1 in resting peripheral bloodmononuclear cells (PBMCs). Expression of all variants is significantlyinduced upon activation of human T cells with anti-CD3 and anti-CD28.The PD-1Δex3 variants lacks a transmembrane domain, and resemblessoluble CTLA-4, which plays an important role in autoimmunity. Ueda etat, Nature 421 506-11 (2003). This variant is enriched in the synovialfluid and sera of patients with rheumatoid arthritis. Wan et al, J.Immunol. 177: 8844-50 (2006). The two PD-1 ligands differ in theirexpression patterns. PD-L1 is constitutively expressed on mouse T and Bcells, CDs, macrophages, mesenchymal stem cells and bone marrow-derivedmast cells. Yamazaki et al, J. Immunol. 169: 5538-45 (2002). PD-L1 isexpressed on a wide range of nonhematopoietic cells (e.g., cornea, lung,vascular epithelium, liver nonpar enchymal cells, mesenchymal stemcells, pancreatic islets, placental synctiotrophoblasts, keratinocytes,etc.) [Keir et al, Annu. Rev. Immunol. 26: 677-704 (2008)], and isupregulated on a number of cell types after activation. Both type I andtype II interferons IFN's) upregulate PD-L1. Eppihimer et al,Microcirculation 9: 133-45 (2002); Schreiner et al, J. Neuroimmunol 155:172-82 (2004). PD-L1 expression in cell lines is decreased when MyD88,TRAF6 and MEK are inhibited. Liu et al, Blood HO: 296-304 (2007). JAK2has also been implicated in PD-L1 induction. Lee et al, FEBS Lett. 580:755-62 (2006); Liu et al, Blood HO: 296-304 (2007). Loss or inhibitionof phosphatase and tensin homolog (PTEN), a cellular phosphatase thatmodified phosphatidylinosital 3-kinase (PI3K) and Akt signaling,increased post-transcriptional PD-L1 expression in cancers. Parse et al,Nat. Med. 13: 84-88 (2007). PD-12 expression is more restricted thanPD-L1. PD-L2 is inducibly expressed on DCs, macrophages, and bonemarrow-derived mast cells. PD-L2 is also expressed on about half totwo-thirds of resting peritoneal BI cells, but not on conventional B2 Bcells. Zhong et al, Eur. J. Immunol. 37: 2405-10 (2007). PD-L2+ BI cellsbind phosphatidylcholine and may be important for innate immuneresponses against bacterial antigens. Induction of PD-L2 by IFN-γ ispartially dependent upon NF-KB. Liang et al, Eur. J. Immunol, 33_:2706-16 (2003). PD-L2 can also be induced on monocytes and macrophagesby GM-CF, IL-4 and and IFN-γ. Yamazaki et al, J. Immunol. 169: 5538-45(2002); Loke et al, PNAS 100:5336-41 (2003).

PDA-1 signaling typically has a greater effect on cytokine productionthan on cellular proliferation, with significant effects on IFN-γ, TNF-αand IL-2 production. PD-1 mediated inhibitory signaling also depends onthe strength of the TCR signaling, with greater inhibition delivered atlow levels of TCR stimulation. This reduction can be overcome bycostimulation through CD28 [Freeman et al, J. Exp. Med. 192: 1027-34(2000)] or the presence of IL-2 [Carter et al, Eur. J. Immunol. 32:634-43 (2002)].

Evidence is mounting that signaling through PD-L1 and PD-L2 may bebidirectional.

That is, in addition to modifying TCR or BCR signaling, signaling mayalso be delivered back to the cells expressing PD-L1 and PD-L2. Whiletreatment of dendritric cells with a naturally human anti-PD-L2 antibodyisolated from a patient with Waldenstrom's macroglobulinemia was notfound to apregulate MHC II or B7 costimulatory molecules, such cells didproduce greater amount of proinflammatory cytokines, particularly TNF-αand IL-6, and stimulated T cell proliferation. Nguyen et al, J. Exp.Med. 196: 1393-98 (2002). Treatment of mice with this antibody also (1)enhanced resistance to transplated bl6 melanoma and rapidly inducedtumor-specific CTL. Radhakrishnan et al, J. Immunol. 170: 1830-38(2003); Radhakrishnan et al, Cancer Res. 64: 4965-72 (2004); Heckman etal, Eur. J. Immunol. 37: 1827-35 (2007); (2) blocked development ofairway inflammatory disease in a mouse model of allergic asthma.Radhakrishnan et al, J. Immunol, 173: 1360-65 (2004); Radhakrishnan etal, J. Allergy Clin. Immunol. UJy. 668-74 (2005).

Further evidence of reverse signaling into dendritic cells (“DDs”)results from studies of bone marrow derived DCs cultured with solublePD-1 (PD-1 EC domain fused to Ig constant region—“s-PD-1”). Kuipers etal, Eur. J. Immunol. 36: 2472-82 (2006). This sPD-1 inhibited DCactivation and increased IL-10 production, in a manner reversiblethrough administration of anti-PD-1. Additionally, several studies showa receptor for PD-L1 or PD-L2 that is independent of PD-1. B7.1 hasalready been identified as a binding partner for PD-L1. Butte et al,Immunity 27: 111-22 (2007). Chemical crosslinking studies suggest thatPD-L1 and B7.1 can interact through their IgV-like domains. B7.1:PD-L1interactions can induce an inhibitory signal into T cells.

Ligation of PD-L1 on CD4+ cells by B7.1 or ligation of B7.1 on CD4+cells by PD-L1 delivers an inhibitory signal. T cells lacking CD28 andCTLA-4 show decreased proliferation and cytokine production whenstimulated by anti-CD3 plus B7.1 coated beads. In T cells lacking allthe receptors for B7.1 (i.e., CD28, CTLA-4 and PD-L1), T-cellproliferation and cytokine production were no longer inhibited byanti-CD3 plus B7.1 coated beads. This indicates that B7.1 actsspecifically through PD-L1 on the T-cell in the absence of CD28 andCTLA-4. Similarly, T cells lacking PD-1 showed decreased proliferationand cytokine production when stimulated in the presence of anti-CD3 plusPD-L1 coated beads, demonstrating the inhibitory effect of PD-L1ligation on B7.1 on T cells. When T cells lacking all known receptorsfor PD-L1 (i.e., no PD-1 and B7.1), T cell proliferation was no longerimpaired by anti-CD3 plus PD-L1 coated beads. Thus, PD-L1 can exert aninhibitory effect on T cells either through B7.1 or PD-1.

The direct interaction between B7.1 and PD-L1 suggests that the currentunderstanding of costimulation is incomplete, and underscores thesignificance to the expression of these molecules on T cells. Studies ofPD-L1^(-/-) T cells indicate that PD-1 on cells can downregulate T cellcytokine production. Latchman et al, Proc. Natl. Acad. Sci. USA 101:10691-96 (2004). Because both PD-L1 and B7.1 are expressed on T cells, Bcells, DCs and macrophages, there is the potential for directionalinteractions between B7.1 and PD-L1 on these cell types. Additionally,PD-L1 on non-hematopoietic cells may interact with B7.1 as well as PD-1on T cells, raising the question of whether PD-L1 is involved in theirregulation. One possible explanation for the inhibitory effect ofB7.1:PD-L1 interaction is that T cell PD-L1 may trap or segregate awayAPC B7.1 from interaction with CD28.

As a result, the antagonism of signaling through PD-L1, includingblocking PD-L1 from interacting with either PD-1, B7.1 or both, therebypreventing PD-L1 from sending a negative co-stimulatory signal toT-cells and other antigen presenting cells is likely to enhance immunityin response to infection (e.g., acute and chronic) and tumor immunity.In addition, the anti-PD-L1 antibodies of the present invention, may becombined with antagonists of other components of PD-1:PD-L1 signaling,for example, antagonist anti-PD-1 and anti-PD-L2 antibodies.

In particular, the inhibition of PD-L1 signaling has been proposed as ameans to enhance T cell immunity for the treatment of cancer (e.g.,tumor immunity) and infection, including both acute and chronic (e.g.,persistent) infection.

Inhibitors blocking the PD-L1:PD-1 interaction are known from, i.a.,WO2001014557, WO2002086083, WO2007005874, WO2010036959, WO2010077634 andWO2011066389. However, as an optimal therapeutic directed to a target inthis pathway has yet to be commercialized, a significant unmet medicalneed exists.

DESCRIPTION OF THE INVENTION

It is an objective of the present invention to provide for anti-PD-L1antibodies, including nucleic acids encoding and compositions containingsuch antibodies, and for their use to enhance T-cell function toupregulate cell-mediated immune responses and for the treatment of Tcell dysfunctional disorders, such as tumor immunity. Surprising, it wasfound that the anti-PD-L1 antibodies according to the present invention,which have antibody dependent cell-mediated cytotoxicity (ADCC)activity, directly act on PD-L1 hearing tumor cells by inducing theirlysis without showing any significant toxicity. Moreover, the antibodiesdo not only block the interaction between human PD-L1 and human PD-L1,but also the interactions between the respective mouse and cynomolgusmonkey proteins.

In one embodiment, the invention provides for an isolated heavy chainvariable region polypeptide comprising an HVR-H1, HVR-H2 and HVR-H3sequence, wherein:

-   (a) the HVR-H1 sequence is X₁YX₂MX₃ (SEQ ID NO:1);-   (b) the HVR-H2 sequence is SIYPSGGX₄TFYADX₅VKG (SEQ ID NO:2);-   (c) the HVR-H3 sequence is IKLGTVTTVX₆Y (SEQ ID NO:3);-   further wherein: X₁ is K, R, T, Q, G, A, W, M, I or S; X₂ is V, R,    K, L, M or I; X₃ is H, T, N, Q, A, V, Y, W, F or M; X₄ is F or I; X₅    is S or T; X₆ is E or D.

In a preferred embodiment X₁ M, I or S; X₂ is R, K, L, M or I; X₃ is For M; X₄ is F or I; X₅ is S or T; X₅ is E or D.

In a more preferred embodiment is X₁ is M, I or S; X₂ is L, M or I; X₃ For M; X₄ is I; X₅ is S or T; X₆ is D.

In a even more preferred embodiment, X₁ is S; X₂ is I; X₃ is M; X₄ is I;X₅ is T; X₆ is D.

In another aspect, the polypeptide further comprises variable regionheavy chain framework sequences juxtaposed between the HVRs according tothe formula:(HC-FR1)-(HVR-H1)-(HC-FR2)-(HVR-H2)-(HC-FR3)-(HVR-H3)-(HC-FR4).

In yet another aspect the frarnework sequences are derived from humanconsensus framework sequences or human germline framework sequences.

In a still further aspect, at least one at the framework sequences isthe following:

(SEQ ID NO: 4) HC-FR1 is EVQLLESGGGLVQPGGSLRLSCAASGFTFS; (SEQ ID NO: 5)HC-FR2 is WVRQAPGKGLEWVS; (SEQ ID NO: 6)HC-FR3 is RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAR; (SEQ ID NO: 7)HC-FR4 is WGQGTLVTVSS.

In a still further aspect, the heavy chain polypeptide is furthercombined with a variable region light chain comprising an an HVR-L1,HVR-L2 and HVR-L3, wherein:

-   (a) the HVR-L1 sequence is TGTX₇X₈DVGX⁹YNYVS (SEQ ID NO:8);-   (b) the HVR-L2 sequence is X₁₀VX₁₁X₁₂RPS (SEQ ID NO:9);-   (c) the HVR-L3 sequence is SSX¹³TX₁₄X₁₅X₁₆X₁₇RV (SEQ ID NO:10);-   further wherein: X₇ is N or S; X₆ is T, R or S; X₉ is A or G; X₁₀ is    E or D; X₁₁ is I, N or S; X₁₂ is D, H or N; X₁₃ is F or Y; X₁₄ is N    or S; X₁₅ is R, T or S; X₁₅ is G or S; X₁₇ is I or T.

In a preferred embodiment, X₇ is N or S; X₈ is T, R or S; X₉ is A or G;X₁₀ is E or D; X₁₁ N or S; X₁₂ is N; X₁₃ is F or Y; X14 is S; X₁₅ is S;X₁₆ is G or S; X₁₇ is T.

In a even more preferred embodiment, X₇ is S; X₈ is S; X₉ is G; X₁₀ isD; X₁₁ is S; X₁₂ is N; X₁₃ is Y; X₁₄ is S; X₁₅ is S; X₁₆ is S; X₁₇ is T.

In a still further aspect, the light chain further comprises variableregion light chain framework sequences juxtaposed between the HVRsaccording to the formula:(LC-FR1)-(HVR-L1)-(LC-FR2)-(HVR-L2)-(LC-FR3)-(HV-L3)-(LC-FR4).

In a still further aspect, the light chain framework sequences arederived from human consensus framework sequences or human germlineframework sequences.

In a still further aspect, the light chain framework sequences arelambda light chain sequences.

In a still further aspect, at least one of the framework sequence is thefollowing:

(SEQ ID NO: 1) LC-FR1 is QSALTQPASVSGSPGQSITISC; (SEQ ID NO: 12)LC-FR2 is WYQQHPGKAPKLMIY; (SEQ ID NO: 13)LC-FR3 is GVSNRFSGSKSGNTASLTISGLQAEDEADYYC; (SEQ ID NO: 14)LC-FR4 is FGTGTKVTVL.

In another embodiment, the invention provides an isolated anti-PD-L1antibody or antigen binding fragment comprising a heavy chain and alight chain variable region sequence, wherein:

-   (a) the heavy chain comprises an HVR-H1, HVR-H2 and HVR-H3, wherein    further; (i) the HVR-H1 sequence is X₁YX₂MX₃ (SEQ ID NO:1); (ii) the    HVR-H2 sequence is SIYPSGGX₄TFYADX₅VKG (SEG ID NO:2); (iii) the    HVR-H3 sequence is IKLGTVTTV6Y, and (SEQ ID NO:3);-   (b) the light chain comprises an HVR-L1, HVR-L2 and HVR-L3, wherein    further: (iv) the HVR-L1 sequence is TGTXX₇X₈DVGX₉YNYVS (SEQ ID    NO:8); (v) the HVR-L2 sequence is X₁₀VX₁₁X₁₂RPS (SEQ ID No:9); (vi)    the HVR-L3 sequence is SSX₁₃TX₁₄X₁₅X₁₆X₁₇RV (SEQ ID NO:10); wherein:    X₁ is K, R, T, Q, G, A, W, M, I or S; X₂ is V, R, K, L, M or I; X₃    is H, T, N, Q, A, V, Y, W, F or M; X₄ is F or I; X₅ is S or T; X₆ is    E or D; X₇ is N or S; X₈ is T, R or S; X₉ is A or G; X₁₀ is E or D;    X₁₁ is I, N or S; X₁₂ is D, H or N; X₁₃ is F or Y; X₁₄ is N or S;    X₁₅ is R, T or S; X₁₆ is G or S; X₁₇ is I or T.

In a preferred embodiment, X₁ is M, I or S; X₂ is R, K, L, M or I; X₃ isF or M; X₄ is F or I; X₅ is S or T; X₆ is E or D; X₇ is N or S; X₈ is T,R or S; X₉ is A or G; X₁₀ is E or D; X₁₁ is N or S; X₁₂ is N; X₁₃ is For Y; X₁₄ is S; X₁₅ is G or S; X₁₇ is T.

In a more preferred embodiment, X₁ is M, I or S; X₂ is L, M or I; X₃ isF or M; X₄ is I; X₅ is S or T; X₆ is D; X₇ is N or S; X₈ is T, R or S;X₉ is A or G; X₁₀ is E or D; X₁₁ is N or S; X₁₂ is N; X₁₃ is F or Y; X₁₄is S; X₁₅ is S; X₁₆ is G or S; X₁₇ is T.

In a even more preferred embodiment, X₁ is S; X₂ is I; X₃ is M; X₄ is I;X₅ is T; X₆ is D; X₇ is S; X₈ is S; X₉ is G; X₁₀ is D; X₁₁ is S; X₁₂ isN; X₁₃ is Y; X₁₄ is S; X₁₅ is S; X₁₆ is S; X₁₇ is T.

In a further aspect, the heavy chain variable region comprises one ormore framework sequences juxtaposed between the HVRs as:(HC-FR1)-(HVR-H1)-(HC-FR2)-(HVR-H2)-(HC-FR3)(HVR-H3)-(HC-FR4), and thelight chain variable regions comprises one or more framework sequencesjuxtaposed between the HVRs as:(LC-FR1)-(HVR-L1)-(LC-FR2)-(HVR-L2)-(LC-FR3)-(HVR-L3)-(LC-FR4).

In a still further aspect, the framework sequences are derived fromhuman consensus framework sequences or human germline sequences.

In a still further aspect one or more of the heavy chain frameworksequences is the following:

(SEQ ID NO: 4) HC-FR1 is EVQLLESGGGLVQPGGSLRLSCAASGFTFS; (SEQ ID NO: 6)HC-FR2 is WVRQAPC3KGLEWVS; (SEQ ID NO: 6)HC-FR3 is RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAR; (SEQ ID NO: 7)HC-FR4 is WGQGTLVTVSS.

In a still further aspect, the light chain framework sequences arelambda light chain sequences.

In a still further aspect, one or more of the light chain frameworksequences is the following:

(SEQ ID NO: 11) LC-FR1 is QSALTDPASVSGSPGQSITISC; (SEQ ID NO: 12)LC-FR2 is WYQQHPGKAPKLMIY; (SED ID NO: 13)LC-FR3 is GVSNRFSGSKSGNTASLTISGLQAEDEADYYC; (SEQ ID NO. 14)LC-FR4 is FGTGTKVTVL.

In a still further aspect, the heavy chain variable region polypeptide,antibody or antibody fragment further comprises at least a C_(H)1domain.

In a more specific aspect, the heavy chain variable region polypeptide,antibody or antibody fragment further comprises a C_(H)1, a CF_(H)2 anda C_(H)3 domain.

In a still further aspect, the variable region light chain, antibody orantibody fragment further comprises a C_(L) domain.

In a still further aspect, the antibody further comprises a C_(H)1, aCF_(H)2 and a C_(H)3 and a C_(L) domain.

In a still further specific aspect, the antibody further comprises ahuman or murine constant region.

In a still further aspect, the human constant region is selected fromthe group consisting of IgG1, IgG2, IgG2, IgG3, IgG4.

In a still further specific aspect, the human or murine constant regionis IgG1.

In yet another embodiment, the invention provides for an anti-PD-L1antibody comprising a heavy chain and a light chain variable regionsequence, wherein:

-   (a) the heavy chain comprises an HVR-H1, HVR-H2 and an HVR-H3,    having at least 80% overall sequence identity to SYIMM (SEQ ID    NO:15), SIYPSGGITFYADTVKG (SEQ ID NO:16) and IKLGTVTTVDY (SEQ ID    NO:17), respectively, and-   (b) the light chain comprises an HVR-L1, HVR-L2 and an HVR-L3,    having at least 80% overall sequence identity to TGTSSDVGGYNVS (SEQ    ID NO:18), DVSNRPS (SEQ ID NO:19) and SSYTSSSTRV (SEQ ID NO:20),    respectively.

In a specific aspect, the sequence identity is 81%, 82%, 83%, 84%, 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or100.

In yet another embodiment, the invention provides for an anti-PD-L1antibody comprising a heavy chain and a light chain variable regionsequence, wherein:

-   (a) the heavy chain comprises an HVR-H1, HVR-H2 and an HVR-H3,    having at least 80% overall sequence identity to MYMMM (SEQ ID    NO:21), SIYPSGGITFYADSVKG (SEQ ID NO:22) and IKLGTVTTVDY (SEQ ID    NO:17), respectively, and-   (b) the light chain comprises an HVR-L1, HVR-L2 and an HVR-L3,    having at least 80% overall sequence identity to TGTSSDVGAYNYVS (SEQ    ID NO:23), DVSNRPS (SEQ ID NO:19) and SSYTSSSTRV (SEQ ID NO:20),    respectively.

In a specific aspect, the sequence identity is 81%, 82%, 83%, 84%, 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 98% 97%, 98%, 99% or100%.

In a still further aspect, in the antibody or antibody fragmentaccording to the invention, as compared to the sequences of HVR-H1 (SEQID NO:15), HVR-H2 (SEQ ID NO:16) and HVV-H3 (SEQ ID NO:17), at leastthose amino acids remain unchanged that are highlighted by underliningas follows:

(SEQ ID NO: 15) (a) in HVR-H1 SYIMM, (SEQ ID NO: 16)(b) in HVR-H2 SIYPSGGITFYADTVKG, (SEQ ID NO: 17)(c) in HVR-H3 IKLGTVTTVDY;and further wherein, as compared to the sequences of HVR-L1 (SEQ IDNO:18), HVR-L2 (SEQ ID NO:19) and HVR-L3 (SEQ ID NO:20) at least thoseamino acids remain unchanged that are highlighted by underlining asfollows:

(SEQ ID NO: 18) (a) HVR-L1 TGTSSDVGGYNYVS (SEQ ID NO: 19)(b) HVR-L2 DVSNRPS (SEQ ID NO: 20) (c) HVR-L3 SSYTSSSTRV.

In another aspect, the heavy chain variable region comprises one or moreframework sequences juxtaposed between the HVRs as:(HC-FR1)-(HVR-H1)-(HC-FR2)-(HVR-H2)-(HC-FR3)(HVR-H3)-(HC-FR4), and thelight chain variable regions comprises one or more framework sequencesjuxtaposed between the HVRs as:(LC-FR1)-(HVR-L1)-(LC-FR2)-(HVR-L2)-(LC-FR3)-(HVR-L3)-(LC-FR4).

In yet another aspect, the framework sequences are derived from humangermline sequences.

In a still further aspect, one or mere of the heavy chain frameworksequences is the following:

(SEQ ID NO: 4) HC-FR1 is EVQLLESGGGLVQPSGGSLRLSCAASGFTFS; (SEQ ID NO: 5)HC-FR2 is WVRQAPGKGLEWVS; (SEQ ID NO: 6)HC-FR3 is RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAR; (SEQ ID NO: 7)HC-FR4 is WGQGTLVTVL.

In a still further aspect, the light chain framework sequences arederived from a lambda light chain sequence.

In a still further aspect, one or more of the light chain frameworksequences is the following:

(SEQ ID NO: 11) LC-FR1 is QSALTQPASVSGSPGQSITISC; (SEQ ID NO: 12)LC-FR2 iS WYQQHPGKAPKLMIY; (SEQ ID NO: 13)LC-FR3 is GVSNRFSGSKSGNTASLTISGLQAEDEADYYC; (SEQ ID NO: 14)LC-FR4 is FGTGTKVTVL.

In a still further specific aspect, the antibody further comprises humanor murine constant region.

In a still further aspect, the human constant region is selected fromthe group consisting of IgG1, IgG2, IgG2, IgG3, IgG4.

In a still further embodiment, the invention provides for an isolatedanti-PD-L1 antibody comprising a heavy chain and a light chain variableregion sequence, wherein:

-   (a) the heavy chain sequence has at least 85% sequence identity to    the heavy chain sequence:

(SEQ ID NO: 24) EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYIMMWVRQAPGKGLEWVSSIYPSGGITFYADTVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARIK LGTVTTVDYWGQGTLVTVSS,and

-   (b) the light chain sequence has at least 85% sequence identity to    the light chain sequence:

(SEQ ID NO: 25) QSALTQPASVSGSPGQSITISCTGTSSDVGGYNWSWYQQHPGKAPKLMIYDVSNRPSGVSNRFSGSKSGNTASLTISGLQAEDEADYYCSSYTSSSTRVF GTGTKVTVL.

In a specific aspect, the sequence identity is 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%.

In a still further embodiment, the invention provides for an isolatedanti-PD-L1 antibody comprising a heavy chain and a light chain variableregion sequence, wherein:

-   (a) the heavy chain sequence has at least 85% sequence identity to    the heavy chain sequence:

(SEQ ID NO: 26) EVQLLESGGSLVQPGGSLRLSCAASGFTFSMYMMMWVRQAPGKGLEWVSSIYPSGGITFYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAIYYCARIKLGTVTTVDYWGQGTLVTVSS, and

-   (b) the light chain sequence has at least 85 sequence identity to    the light chain sequence:

(SEQ ID NO: 27) QSALTQPASVSGSPGRSITISCTGTSSDVGAYNYVSWYOQHPGKAPKLMIYDVSNRPSGVSNRFSGSKSGNTASLTISGLQAEDEADYYCSSYTSSSTRV FGTGTKVTVL.

In a specific aspect, the sequence identity is 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%.

In another embodiment the antibody binds to human, mouse or cynomolgusmonkey PD-L1. In a specific aspect the antibody is capable of blockingthe interaction between human, mouse or cynomolgus monkey PD-L1 and therespective human, mouse or cynomolgus monkey PD-1 receptors.

In another embodiment, the antibody binds to human PD-L1 with a K_(D) of5×10⁻⁹ M or less, preferably with a K_(D) of 2×10⁻⁹ M or less, and evenmore preferred with a K_(D) of 1×10⁻⁹ M or lees.

In yet another embodiment the invention concerns an isolated anti-PD-L1antibody or antigen binding fragment thereof which binds to a functionalepitope comprising residues Y56 and D61 of human PD-L1 (SEQ ID NO28).

In a specific aspect, the functional epitope further comprises E58, E60,Q68, R113 and M115 of human PD-L1 (SEQ. ID NO:28).

In a more specific aspect, the antibody binds to a conformationalepitope, comprising residues 54-60 and 112-122 of human PD-L1 (SEQ IDNO:28).

In a further embodiment, the invention is related to an anti-PD-L1antibody, or antigen binding fragment thereof, which cross-competes forbinding to PD-L1 with an antibody according to the invention asdescribed herein.

In a still further embodiment, the invention provides for compositionscomprising any of the above described anti-PD-L1 antibodies incombination with at least one pharmaceutically acceptable carrier.

In a still further embodiment, the invention provides for an isolatednucleic acid encoding a polypeptide, or light chain or a heavy chainvariable region sequence of an anti-PD-L1 antibody, or antigen bindingfragment thereof, as described herein. In a still further embodiment,the invention provides for an isolated nucleic acid encoding a lightchain or a heavy chain variable region sequence of an anti-PD-L1antibody, wherein:

-   (a) the heavy chain comprises a HVR-H1, HVR-H2 and an HVR-H3    sequence having at least 80% sequence identity to SYIMM (SEQ ID    NO:15), SIYPSGGITFYADTVKG (SEQ ID NO:16) and IKLGTVTTVDY (SEQ ID NO:    17), respectively, of-   (b) the light chain comprises an HVR-1, HVR-L2 and an HVR-L3    sequence having at least 80% sequence identity to TGTSSDVGGYNYVS    (SEQ ID NO:18), DVSNRPS (SEQ ID NO:19) and SSYTSSSTRV (SEQ ID    NO:20), respectively.

In a specific aspect, the sequence identity is 81%, 82%, 83%, 84%, 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or100%.

In a further aspect the nucleic acid is SEQ ID NO:30 for the heavychain, and SEQ ID NO:31 for the light chain.

In another aspect, the nucleic acid further comprises a vector suitablefor expression of the nucleic acid encoding any of the previouslydescribed anti-PD-L1 antibodies.

In a still further specific aspect, the vector further comprises a hostcell suitable for expression of the nucleic acid.

In a still further specific aspect, the host cell is a eukaryotic cellor a prokaryotic cell.

In a still further specific aspect, the eukaryotic cell is a mammaliancell, such as Chinese Hamster Ovary (CHO).

In a still further embodiment, the invention provides for a process ofmaking an anti-PD-L1 antibody or antigen binding fragment thereof,comprising culturing a host cell containing nucleic acid encoding any ofthe previously described anti-PD-L1 antibodies or antigen-bindingfragment in a form suitable for expression, under conditions suitable toproduce such antibody or fragment, and recovering the antibody orfragment.

In a still further embodiment, the invention provides a kit of partscomprising a container enclosing a therapeutically effective amount of acomposition disclosed herein and a package insert indicating use for thetreatment of a T-cell dysfunctional disorder.

In a still further embodiment the invention provides for a kit of partscomprising any of the above described anti-PD-L1 compositions incombination with at least one further therapeutic agent or vaccine, suchas a chemotherapeutic agent.

In one aspect the at least one chemotherapeutic agent is gemcitabine,cyclophosphamide, fluorouracil or oxaliplatin.

In another aspect, the vaccine is Stimuvax.

In a still further embodiment, the invention provides for a method ofenhancing T-cell function comprising administering an effective amountof any of the above described anti-PD-L1 antibodies or compositions.

In one aspect, the anti-PD-L1 antibody or composition rendersdysfunctional T-cells non-dysfunctional.

In another aspect, the antibody or composition treats of prevents asymptom of persistent infection, such as viral infection, e.g. by humanimmunodeficiency virus (HIV), herpes virus, Eppstein-Barr virus or humanpapilloma virus.

In a still further embodiment, the invention provides for a method oftreating a T-cell dysfunctional disorder comprising administering atherapeutically effective amount of any of the above describedanti-PD-L1 antibodies or compositions.

In one specific aspect, the T-cell dysfunctional disorder is tumorimmunity.

In a still further aspect, the method further comprises treatment with avaccine.

In a still further aspect, the PD-L1 antibody or composition is combinedwith a treatment regimen further comprising a traditional therapyselected from the group consisting of: surgery, radiation therapy,chemotherapy, targeted therapy, immunotherapy, hormonal therapy,angiogenesis inhibition and palliative care.

In a still further specific aspect, the tumor immunity results from acancer selected from the group consisting of: breast, lung, colon,ovarian, melanoma, bladder, kidney, liver, salivary, stomach, gliomas,thyroid, thymic, epithelial, head and neck cancers, gastric, andpancreatic cancer.

Another aspect of the invention relates to the use of antibody dependentcell-mediated cytotoxicity (ADCC) of an anti-PD-L1 antibody disclosedherein or composition in the treatment of cancer.

Therefore, the invention pertains to method of treating cancercomprising administering to a subject in need thereof an effectiveamount of an anti-PD-L1 antibody which induces antibody dependentcell-mediated cytotoxicity (ADCC).

In a preferred embodiment the constant region of the anti-PD-L1 antibodyis IgG1.

In another preferred embodiment the cancer is selected from the groupconsisting of: breast, lung, colon, ovarian, melanoma, bladder, kidney,liver, salivary, stomach, gliomas, thyroid, thymic, epithelial, head andneck cancers, gastric and pancreatic cancer.

Equivalent to the above mentioned methods of enhancing T-cell function,treating a T-cell dysfunctional disorder, or treating cancer, theinvention relates likewise to the use of an anti-PD-L1 antibody orcomposition as described above and below for the manufacture at amedicament for chancing T-cell function, treating a T-cell dysfunctionaldisorder or treating cancer;

or to an anti-PD-L1 antibody or composition for use in the enhancementof T-cell function, or treatment of a T-cell dysfunctional disorder orcancer.

In yet a further embodiment, the invention is directed to engineeredantibodies, or engineered antibody fragments, which are fused directlyor via a linker molecule to therapeutic agents, such as cytokines (e.g.IL-2, IL-12, TNFa, IFNa, IFNb), or growth factors; which engineeredantibodies or engineered antibody fragments may also be used in tumortherapy and immune system related diseases. Antibody fusion proteins,especially immunocytokines, are well known in the art. The fusionpartner can be bound to the N-terminus of the antibody or antibodyfragment or, preferably, to its C-terminus.

Definitions

“Dysfunction” in the context of immune dysfunction, refers to a state ofimmune reduced responsiveness to antigenic stimulation. The termincludes the common elements of both exhaustion and/or anergy in whichantigen recognition may occur, but the ensuing immune response isineffective to control infection or tumor growth.

“Enhancing T-cell function” means to induce, cause or stimulate a T-cellto have a sustained or amplified biological function, or renew orreactivate exhausted or inactive T-cells. Examples of enhancing T-cellfunction include: increased secretion of γ-interferon from CD8⁺ T-cells, increased proliferation, increased antigen responsiveness (e.g.,viral or pathogen clearance) relative to such levels before theintervention. In one embodiment, the level of enhancement is as least50%, alternatively 60%, 70%, 80%, 90%, 100%, 120%, 150%, 200%. Themanner of measuring this enhancement is known to one of ordinary skillin the art.

A “T cell dysfunctional disorder” is a disorder or condition of T-cellscharacterized by decreased responsiveness to antigenic stimulation. In aparticular embodiment a T-cell dysfunctional disorder is a disorder thatis specifically associated with inappropriate increased signalingthrough PD-1. In another embodiment, T-cell dysfunctional disorder isone in which T-cells are anergic or have decreased ability to secretecytokines, proliferate, or execute cytolytic activity. In a specificaspect, the decreased responsiveness results in ineffective control of apathogen or tumor expressing an immunogen. Examples of T celldysfunctional disorders characterized by T-cell dysfunction includeunresolved acute infection, chronic infection and tumor immunity.

“Tumor immunity” refers to the process in which tumors evade immunerecognition and clearance. Thus, as a therapeutic concept, tumorimmunity is “treated” when such evasion is attenuated, and the tumorsare recognized and attacked by the immune system. Examples of tumorrecognition include tumor binding, tumor shrinkage and tumor clearance.

The term “vaccine” as used herein includes any nonpathogenic immunogenthat, when inoculated into a host, induces protective immunity against aspecific pathogen. Vaccines can take many forms. Vaccines can be wholeorganisms that share important antigens with the pathogen, but are notpathogenic themselves (e.g., cowpox). Vaccines can also be prepared fromkilled (e.g., Salk polio vaccine) or attenuated (lost ability to producedisease—e.g., Sabin polio vaccine). Vaccines can also be prepared frompurified macromolecules isolated from the pathogenic organism. Forexample, toxoid vaccines (e.g., tetanus and diphthena) containing theinactive form of soluble bacterial toxin—resulting in the production ofanti-toxin antibodies, but not immunity to the intact bacteria. Subunitvaccines (e.g., Hepatitis B) contain only a single immunogenic proteinisolated from the pathogen of interest. Hapten conjugate vaccinesattaches certain carbohydrate or polypeptide epitopes isolated from thepathogen of interest to immunogenic carriers, such as tetanus toxoid.These strategies essentially use the epitopes as haptens to induceantibody production, which then recognize the same epitope in the nativepathogen. However, to be maximally effective, such vaccines mustincorporate both B- and T-cell cell epitopes, and the T-cell epitopesmust be chosen to ensure that they can be recognized, presented andresponded to by the immune systems of the host individuals. DNA vaccinesexploit the ability of host cells to take up and express DNA encodingpathogenic proteins that is injected intramuscularly. Host responses toimmunogens can be enhanced if administered as a mixture with adjuvants.Immune adjuvants function in one or more of the following ways: (1)prolonging retention of the immunogen, (2) increased effective size ofthe immunogen (and hence promoting phagocytosis and presentation tomacrophages), (3) stimulating the influx of macrophage or other immunecells to the injection site, or (4) promoting local cytokine productionand other immunologic activities. Example adjuvants include: completeFreund's adjuvant (CFA), aluminum salts, and mycobacterial derivedproteins such as muramyl di- or tri-peptides.

The term “antibody” includes monoclonal antibodies (including fulllength antibodies which have an immunoglobulin Fc region), antibodycompositions with polyepitopic specificity, multispecific antibodies{e.g., bispecific antibodies, diabodies, and single-chain molecules, aswell as antibody fragments (e.g., Fab, F(ab′)₂, and Fv). The term“immunoglobulin” (Ig) is used interchangeably with “antibody” herein.The basic 4-chain antibody unit is a heterotetrameric glycoproteincomposed of two identical light (L) chains and two identical heavy (H)chains. An IgM antibody consists of 5 of the basic heterotetramer unitsalong with an additional polypeptide called a J chain, and contains 10antigen binding sites, while IgA antibodies comprise from 2-5 of thebasic 4-chain units which can polymerize to form polyvalent assemblagesin combination with the J chain. In the case of IgGs, the 4-chain unitis generally about 150,000 daltons. Each L chain is linked to an H chainby one covalent disulfide bond, while the two H chains are linked toeach other by one or more disulfide bonds depending on the H chainisotype. Each H and L chain also has regularly spaced intrachaindisulfide bridges. Each H chain has at the N-terminus, a variable domain(V_(H)) followed by three constant domains (C_(H)) for each of the α andγ chains and four C_(H) domains for μ and ε isotypes. Each L chain hasat the N-terminus, a variable domain (V_(L)) followed by a constantdomain at its other end. The V_(L) is aligned with the V_(H) and theC_(L) is aligned with the first constant domain of the heavy chain(C_(H)1). Particular amino acid residues are believed to form aninterface between the light chain and heavy chain variable domains. Thepairing of a V_(H) and V_(L) together forms a single antigen-bindingsite. For the structure and properties of the different classes ofantibodies, see e.g., Basic and Clinical Immunology, 8th Edition, DanielP. Sties, Abba I. Terr and Tristram G. Parsolw (eds), Appleton & Lange,Norwalk, Conn., 1994, page 71 and Chapter 6. The L chain from anyvertebrate species can be assigned to one of two dearly distinct types,called kappa and lambda, based on the amino acid sequences of theirconstant domains. Depending on the amino acid sequence of the constantdomain of their heavy chains (CH), immunoglobulins can be assigned todifferent classes or isotypes. There are five classes ofimmunoglobulins: IgA, IgD, IgE, IgG and IgM, having heavy chainsdesignated α, δ, ε, γ and μ, respectively. The γ and α classes arefurther divided into subclasses on the basis of relatively minordifferences in the CH sequence and function, e.g., humans express thefollowing subclasses: IgG1, IgG2A, IgG2B, IgG3, IgG4, IgA1 and IgK1.

An “isolated” antibody is one that has been identified, separated and/orrecovered from a component of its production environment (E.g., naturalor recombinant). Preferably, the isolated polypeptide is free ofassociation with all other components from its production environment.Contaminant components of its production environment, such as thatresulting from recombinant transfected cells, are materials that wouldtypically interfere with research, diagnostic or therapeutic uses forthe antibody, and may include enzymes, hormones, and other proteinaceousor non-proteinaceous solutes. In preferred embodiments, the polypeptidewill be purified: (1) to greater than 95% by weight of antibody asdetermined by, for example, the Lowry method, and in some embodiments,to greater than 99% by weight; (1) to a degree sufficient to obtain atleast 15 residues of N-terminal or internal amino acid sequence by useof a spinning cup sequenator, or (3) to homogeneity by SDS-PAGE undernon-reducing or reducing conditions using Coomassie blue or, preferably,silver stain. Isolated antibody includes the antibody in situ withinrecombinant cells since at least one component of the antibody's naturalenvironment will not be present. Ordinarily, however, an isolatedpolypeptide or antibody will be prepared by at least one purificationstep.

The “variable region” or “variable domain” of an antibody refers to theamino-terminal domains of the heavy or light chain of the antibody. Thevariable domains of the heavy rain and light chain may be referred to as“VH” and “VL”, respectively. These domains are generally the mostvariable parts of the antibody (relative to other antibodies of the sameclass) and contain the antigen binding sites.

The term “variable” refers to the fact that certain segments of thevariable domains differ extensively in sequence among antibodies. The Vdomain mediates antigen binding and defines the specificity of aparticular antibody for its particular antigen. However, the variabilityis not evenly distributed across the entire span of the variabledomains. Instead, it is concentrated in three segments calledhypervariable regions (HVRs) both in the light-chain and the heavy chainvariable domains. The more highly conserved portions of variable domainsare called the framework regions (FR). The variable domains of nativeheavy and light chains each comprise four FR regions, largely adopting abeta-sheet configuration, connected by three HVRs, which form loopsconnecting, an in some cases forming part of, the beta-sheet structure.The HVRs in each chain are held together in close proximity by the FRregions and, with the HVRs from the other chain, contribute to theformation of the antigen binding site of antibodies (see Kabat et al,Sequences of Immunological Interest, Fifth Edition, National Instituteof Health, Bethesda, Md. (1991)). The constant domains are not involveddirectly in the binding of antibody to an antigen, but exhibit variouseffector functions, such as participation of the antibody inantibody-dependent cellular toxicity.

The term “monoclonal antibody” as used herein refers to an antibodyobtained from a population of substantially homogeneous antibodies,i.e., the individual antibodies comprising the population are identicalexcept for possible naturally occurring mutations and/orpost-translation modifications (e.g., isomerizations, amidations) thatmay be present in minor amounts. Monoclonal antibodies are highlyspecific, being directed against a single antigenic site. In contrast topolyclonal antibody preparations which typically include differentantibodies directed against different delamlinants (epitopes), eachmonoclonal antibody is directed against a single determinant on theantigen. In addition to their specificity, the monoclonal antibodies areadvantageous in that they are synthesized by the hybridoma culture,uncontaminated by other immunoglobulins. The modifier “monoclonal”indicates the character of the antibody as being obtained from asubstantially homogeneous population of antibodies, and is not to beconstrued as requiring production of the antibody by any particularmethod. For example, the monoclonal antibodies to be used in accordancewith the present invention may be made by a variety of techniques,including, for example, the hybridoma method (e.g., Kohler and Milstein,Nature, 256:495-97 (1975); Hongo et al, Hybridoma, 14 (3): 253-280(1995). Harlow et al, Antibodies: A Laboratory Manual, (Cold SpringHarbor Laboratory Press, 2^(nd) ed. 1988); Hammerling et al, in:Monoclonal Antibodies and T-Cell Hybridomas 563-681 (Elsevier, N.Y.,1981)), recombinant DNA methods (see, e.g., U.S. Pat. No. 4,816,567),phage-display technologies (see, e.g., Clackson et al, Nature, 352:624-628 (1991); Marks et al, J. Mol Biol. 222: 581-597 (1992); Sidhu etal, J. Mol Biol. 338(2):299-310 (2004); Lee et al, J. Mol Biol. 340(5):1073-1093 (2004); Fellouse, Proc. Natl. Acad. ScL USA 101(34):12467-12472 (2004); and Lee et al, J. Immunol. Methods 284(1-2): 119-132(20041), and technologies for producing human or humanlike antibodies inanimals that have parts or all of the human immunoglobulin loci or genesencoding human immunoglobulin sequences (see, e.g., WO 1998/24893; WO1996/34096; WO 1996/33735; WO 1991/10741; Jakobovits et al, Proc. Natl.Acad. ScL USA 90; 2551 (1993); Jakobovits et al, Nature 362: 255-258(1993); Bruggemann et al, Year in Immunol. 7:33 (1993); U.S. Pat. Nos.5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; and 5,661,016;Marks et al, Bio/Technology 10: 779-783 (1992); Lonberg et al, Nature368: 856-859 (1994); Morrison, Nature 368: 812-813 (1994); Fishwild etal, Nature Biotechnol 14: 845-851 (1996); Neuberger, Nature Biotechnol,14: 826 (1996); and Lonberg and Huszar, Intern. Rev. Immunol. 13: 65-93(1995).

“antibody fragment” comprises a portion of an intact antibody,preferably the antigen binding and/or the variable region of the intactantibody. Examples of antibody fragments include Fab, Fab′, F(ab′)₂ andFv fragments; diabodies; linear antibodies (see U.S. Pat. No. 5,641,870,Example 2; Zapata et al, Protein Eng. 8HO): 1057-1062 [1995]);single-chain antibody molecules and multispecific antibodies formed fromantibody fragments. Papain digestion of antibodies produced twoidentical antigen-binding fragments, called “Fab” fragments, and aresidual “Fc” fragment, a designation reflecting the ability tocrystallize readily. The Fab fragment consists of an entire L chainalong with the variable region domain of the H chain (V_(H)), and thefirst constant domain of one heavy chain (C_(H)1). Each Fab fragment ismonovalent with respect to antigen binding, i.e., it has a singleantigen-binding site. Pepsin treatment of an antibody yields a singlelarge F(ab′)₂ fragment which roughly corresponds to two disulfide linkedFab fragments having different antigen-binding activity and is stillcapable of cross-linking antigen. Fab′ fragments differ from Fabfragments by having a few additional residues at the carboxy terminus ofthe C_(H)1 domain including one or more cysteines from the antibodyhinge region. Fab′-SH is the designation herein for Fab′ in which thecysteine residue(s) of the constant domains bear a free thiol group.F(ab′)₂ antibody fragments originally were produced as pairs of Fab′fragments which have hinge cysteines between them. Other chemicalcouplings of antibody fragments are also known.

The Fc fragment comprises the carboxy-terminal portions of both H chainsheld together by disulfides. The effector functions of antibodies aredetermined by sequences in the Fc region, the region which is alsorecognized by Fc receptors (FcR) found on certain types of cells.

“Fv” is the minimum antibody fragment which contains a completeantigen-recognition and -binding site. This fragment consists of a dimerof one heavy- and one light-chain variable region domain in tight,non-covalent association. From the folding of these two domains emanatesix hypervariabie loops (3 loops each from the H and L chain) thatcontribute the amino acid residues for antigen binding and conferantigen binding specificity to the antibody. However, even a singlevariable domain (or half of an Fv comprising only three HVRs specificfor an antigen) has the ability to recognize and bind antigen, althoughat a lower affinity than the entire binding site. “Single-chain Fv” alsoabbreviated as “sFv” or “scFv” are antibody fragments that comprise theV_(H) and V_(L) antibody domains connected into a single polypeptidechain. Preferably, the sFv polypeptide further comprises a polypeptidelinker between the V_(H) and V_(L) domains which enables the sFv to formthe desired structure for antigen binding. For a review of the sFv, seePluckthun in The Pharmacology of Monoclonal Antibodies vol. 113,Rosenburg, and Moore eds., Springer-Verlag, New York, pp. 269-315(1994). “Functional fragments” of the antibodies of the inventioncomprise a portion of an intact antibody, generally including theantigen binding or variable region of the intact antibody or the Fcregion of an antibody which retains or has modified FcR bindingcapability. Examples of antibody fragments include linear antibody,single-chain antibody molecules and multispecific antibodies formed fromantibody fragments.

The term “diabodies” refers to small antibody fragments prepared byconstructing sFv fragments (see preceding paragraph) with short linkers(about 5-10) residues) between the V_(H) and V_(L) domains such thatinter-chain but not intra-chain pairing of the V domains is achieved,thereby resulting in a bivalent fragment, i.e., a fragment having twoantigen-binding sites. Bispecific diabodies are heterodimers of two“crossover” sFv fragments in which the V_(H) and V_(L) domains of thetwo antibodies are present on different polypeptide chains. Diabodiesare described in greater detail in, for example, EP 404,097; WO93/11161, Hollinger et al, Proc. Natl. Acad. ScL USA 90: 6444-6448(1993).

The term “nanobodies” refers to single-domain antibodies which areantibody fragments consisting of a single monomeric variable antibodydomain. Like a whole antibody, they are able to bind selectively to aspecific antigen. With a molecular weight of only 12-15 kDa,single-domain antibodies are much smaller than common antibodies(150-160 kDa). The first single-domain antibodies were engineered fromheavy-chain antibodies found in camelids. Gibbs, W. Wayt (August 2005).“Nanobodies”. Scientific American Magazine.

The monoclonal antibodies herein specifically include “chimeric”antibodies (immunoglobulins) in which a portion of the heavy and/orlight chain is identical with or homologous to corresponding sequencesin antibodies derived from a particular species or belonging to aparticular antibody class or subclass, while the remainder of thechain(s) is(are) identical with or homologous to corresponding sequencesin antibodies derived from another species or belonging to anotherantibody class or subclass, as well as fragments of such antibodies, solong as they exhibit the desired biological activity (U.S. Pat. No.4,816,567; Morrison et al, Proc. Natl. Acad. ScL USA, 81:6851-6855(1984)). As used herein, “humanized antibody” is used a subset of“chimeric antibodies.”

“Humanized” forms of non-human (e.g., murine) antibodies are chimericantibodies that contain minimal sequence derived from non-humanimmunoglobulin. In one embodiment, a humanized antibody is a humanimmunoglobulin (recipient antibody) in which residues from an HVR(hereinafter defined) of the recipient are replaced by residues from anHVR of a non-species (donor antibody) such as mouse, rat, rabbit ornon-human primate having the desired specificity, affinity, and/orcapacity. In some instances, framework (“FR”) residues of the humanimmunoglobulin are replaced by corresponding non-human residues.Furthermore, humanized antibodies may comprise residues that are notfound in the recipient antibody or in the donor antibody. Thesemodifications may be made to further refine antibody performance, suchas binding affinity. In general, a humanized antibody will comprisesubstantially all of at least one, and typically two, variable domains,in which all or substantially all of the hypervariable loops correspondto those of a non-human immunoglobulin sequence, and all orsubstantially all of the FR regions are those of a human immunoglobulinsequence, although the FR regions may include one or more individual FRresidue substitutions that improve antibody performance, such as bindingaffinity, isomerization, immunogenicity, etc. The number of these aminoacid substitutions in the FR are typically no more than 6 in the Hchain, and in the L chain, no more than 3. The humanized antibodyoptionally will also comprise at least a portion of an immunoglobulinconstant region (Fc), typically that of a human immunoglobulin. Forfurther details, see, e.g., Jones et al, Nature 321:522-525 (1986);Riechmann et al, Nature 332:323-329 (1958); and Pesta, Curr. Op. Struct.Biol. 2:593-598 (1992). See also, for example, Vaswani and Hamilton,Ann. Allergy, Asthma & Immunol. 1:105-115 (1998); Harris, Biochem, Soc.Transactions 23:1035-1038 (1995); Hurle and Gross, Curr. Op. Biotech.5:428-433 (1994); and U.S. Pat. Nos. 6,982,321 and 7,087,409.

A “human antibody” is an antibody that possesses an amino-acid sequencecorresponding to that of an antibody produced by a human and/or has beenmade using any of the techniques for making human antibodies asdisclosed herein. This definition of a human antibody specificallyexcludes a humanized antibody comprising non-human antigen-bindingresidues. Human antibodies can be produced using various techniquesknown in the art, including phage-display libraries. Hoogenboom andWinter, J. Mol. Biol, 227:381 (1991); Marks et al, J. Mol. Biol, 222:581(1991). Also available for the preparation of human monoclonalantibodies are Methods described in Cole et al, Monoclonal Antibodiesand Cancer Therapy, Alan R. Liss, p. 77 (1985); Boerner et al, J.Immunol, 147(I):86-95 (1991). See also van Dijk and van de Winkel, Curr.Opin. Pharmacol, 5: 368-74 (2001). Human antibodies can be prepared byadministering the antigen to a transgenic animal that has been modifiedto produce such antibodies in response to antigenic challenge, but whoseendogenous loci have been disabled, e.g., immunized xenomice (see, e.g.,U.S. Pat. Nos. 6,075,181 and 6,150,584 regarding XENOMOUSE™ technology).See also, for example, Li et al, Proc. Natl. Acad. Sci. USA,103:3557-3562 (2006) regarding human antibodies generated via a humanC-cell hybridoma technology.

The term “hypervariable region,” “HVR,” or “HV,” when used herein refersto the regions of an antibody variable domain which are hypervariable insequence and/or form structurally defined loops. Generally, antibodiescomprise six HVRs; three in the VH (H1, H2, H3), and three in the VL(L1, L2, L3). In native antibodies, H3 and L3 display the most diversityof the six HVRs, and H3 in particular is believed to play a unique rolein conferring fine specificity to antibodies. See, e.g., Xu et al,Immunity 13:37-45 (2000); Johnson and Wu, in Methods in MolecularBiology 248:1-25 (Lo, ed., Human Press, Totowa, N.J., 2003). Indeed,naturally occurring camelid antibodies consisting of a heavy chain onlyare functional and stable in the absence of light chain. See, e.g.,Hamers-Casterman et al., Nature 363;446-448 (1993); Sheriff et al,Nature Struct. Biol. 3:733-736 (1996).

A number of HVR delineations are in use and are encompassed herein. TheKabat Complementarity Determining Regions (CDRs) are based on sequencevariability and are the most commonly used (Kabat et al., Sequences ofProteins of Immunological Interest, 5th Ed. Public Health Service,National Institutes of Health, Bethesda, Md. (1991)). Chothia refersinstead to the location of the structural loops (Chothia and Lesk, J.Mol. Biol. 196:901-917 (1987)). The AbM HVRs represent a compromisebetween the Kabat HVRs and Chothia structural loops, and are used byOxford Molecular's AbM antibody modeling software.

The “contact” HVRs are based on an analysis of the available complexcrystal structures. The residues from each of these HVRs are notedbelow.

Loop Kabat AbM Chothia Contact L1 L24-L34 L24-L34 L26-L32 L30-L36 L2L50-L56 L50-L56 L50-L52 L46-L55 L3 L89-L97 L89-L97 L91-L96 L89-L96 H1H31-H35B H26-H35B H26-H32 H30-H35B (Kabat Numbering) H1 H31-H35 H26-H35H26-H32 H30-H35 (Chothia Numbering) H2 H50-H65 H50-H58 H53-H55 H47-H58H3 H95-H102 H95-H102 H96-H101 H93-H101

HVRs may comprise “extended HVRs” follows: 24-36 or 24-34 (L1), 46-56 or50-56 (L2) and 89-97 or 89-96 (L3) in the VL and 26-35 (H1), 50-65 or49-65 (H2) and 93-102, 94-102, or 95-102 (H3) in the VH. The variabledomain residues are numbered according to Kabat et al., supra, for eachof these definitions.

The expression “variable-domain residue-numbering as in Kabat” or“amino-acid-position numbering as in Kabat,” and variations thereof,refers to the numbering system used for heavy-chain variable domains orlight-chain variable domains of the compilation of antibodies in Kabatet al., supra. Using this numbering system, the actual linear amino acidsequence may contain fewer or additional amino acids corresponding to ashortening of, or insertion into, a FR or HVR of the variable domain.For example, a heavy-chain variable domain may include a single aminoadd insert (residue 52a according to Kabat) after residue 52 of H2 andinserted residues (e.g. residues 82a, 82b, and 82c, etc. according toKabat) after heavy-chain FR residue 82. The Kabat numbering of residuesmay be determined for a given antibody by alignment at regions ofhomology of the sequence of the antibody with a “standard” Kabatnumbered sequence.

“Framework” or “FR” residues are those variable-domain residues otherthan the HVR residues as herein defined. A “human consensus framework”or “acceptor human framework” is a framework that represents the mostcommonly occurring amino acid residues in a selection of humanimmunoglobulin VL or VH framework sequences. Generally, the selection ofhuman immunoglobulin VL or VH sequences is from a subgroup of variabledomain sequences. Generally, the subgroup of sequences is a subgroup asin Kabat et al., Sequences of Proteins of Immunological Interest 5^(th)Ed. Public Health Service, National Institutes of Health, Bethesda, Md.(1991). Examples include for the VL, the subgroup may be subgroup kappaI, kappa II, kappa III or kappa IV as in Kabat et al, supra.Additionally, for the VH, the subgroup may be subgroup I, subgroup II,or subgroup III as in Kabat et al., supra. Alternatively, a humanconsensus framework can be derived from the above in which particularresidues, such as when a human framework residue is selected based onits homology to the donor framework by aligning the donor frameworksequence with a collection of various human framework sequences. Anacceptor human framework “derived from” a human immunoglobulin frameworkor a human consensus framework may comprise the same amino acid sequencethereof, or it may contain preexisting amino acid sequence changes. Insome embodiments, the number of pre-existing amino acid changes are 10or less, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 orless, 3 or less, or 2 or less.

“amino-acid modification” at a specified position, e.g. of the Fcregion, refers to the substitution or deletion of the specified residue,or the insertion of at least one amino acid residue adjacent thespecified residue. Insertion “adjacent” to a specified residue meansinsertion within one to two residues thereof. The insertion may beN-terminal or C-terminal to the specified residue. The preferred aminoacid modification herein is a substitution.

An “affinity-matured” antibody is one with one or more alterations inone or more HVRs thereof that result in an improvement in the affinityof the antibody for antigen, compared to a parent antibody that does notpossess those alteration(s). In one embodiment, an affinity-maturedantibody has nanomolar or even picomolar affinities for the targetantigen. Affinity-matured antibodies are produced by procedures known inthe art. For example, Marks et al, Bio/Technology 10:779-783 (1992)describes affinity maturation by VH- and VL-domain shuffling. Randommutagenesis HVR and/or framework residues is described by, for example:Barbas et al. Proc Nat. Acad. ScL USA 91:3809-3813 (1994); Schier et al.Gene 169:147-155 (1995); Y{acute over (α)}Xoη et al. J. Immunol.155:1994-2004 (1995); Jackson et al, J. Immunol. 154(7):3310-9 (1995);and Hawkins et al, J. Mol. Biol. 226:889-896 (1992).

As use herein, the term “specifically binds to” or is “specific for”refers to measurable and reproducible interactions such as bindingbetween a target and an antibody, which is determinative of the presenceof the target in the presence of a heterogeneous population of moleculesincluding biological molecules. For example, an antibody thatspecifically binds to a target (which can be an epitope) is an antibodythat binds this target with greater affinity, avidity, more readily,and/or with greater duration than it binds to other targets. In oneembodiment, the extent of binding of an antibody to an unrelated targetis less than about 10% of the binding of the antibody to the target asmeasured, e.g., by a radioimmunoassay (RIA). In certain embodiments, anantibody that specifically binds to a target has a dissociation constant(K_(D)) of <1×10⁻⁶M, <1×10⁻⁷M, <1×10⁻⁸M, <1× ⁻⁹M, or <1×10⁻¹⁰M. Incertain embodiments, an antibody specifically binds to an epitope on aprotein that is conserved among the protein from different species. Inanother embodiment, specific binding can include, but does not requireexclusive binding.

“Antibody-dependent cell-mediated cytotoxicity” or ADCC refers to a formof cytotoxicity in which secreted Ig bound onto Fc receptors (FcRs)present on certain cytotoxic cells (e.g., natural killer (NK) cells,neutrophils and macrophages) enable these cytotoxic effector cells tobind specifically to an antigen-bearing target cell and subsequentlykill the target cell with cytotoxins. The antibodies “arm” the cytotoxiccells and are required for killing of the target cell by this mechanism.The primary cells for mediating ADCC, NK cells, express FcγRIII only,whereas monocytes express FcγRI, FcγRII and FcγRIII. Fc expression onhematopoietic cells is summarized in Table 3 on page 464 of Ravetch andKinet, Annu. Rev. Immunol. 9: 457-92 (1991). To assess ADCC activity ofa molecule of interest, an in vitro ADCC assay, such as that describedin U.S. Pat. No. 5,500,362 or 5,821,337 may be performed. Usefuleffector cells for such assays include peripheral blood mononuclearcells (PBMC) and natural killer (NK) cells. Alternatively, oradditionally, ADCC activity of the molecule of interest may be assessedin vivo, e.g., in an animal model such as that disclosed in Clynes etal, PNAS USA 95:652-656 (1998). Unless indicated otherwise herein, thenumbering of the residues in an immunoglobulin heavy chain is that ofthe EU index as in Kabat et al, supra. The “EU index as in Kabat” refersto the residue numbering of the human IgG1 EU antibody. In many cancersthe tumor cells express high levels of PD-L1 on their surface. Uponbinding to PD-L1 on tumor cells and binding with their fragmentcrystalline (Fc) part to Fc-gamma receptors (FCGR) leukocytes,anti-PD-L1 antibodies with ADCC potential can trigger ADCC which maylead to the death of these tumor cells.

The term “Fc region” herein is used to define a C-terminal region of animmunoglobulin heavy chain, including native-sequence Fc regions andvariant Fc regions. Although the boundaries of the Fc region of animmunoglobulin heavy chain might vary, the human IgG heavy-chain Fcregion is usually defined to stretch from an amino acid residue atposition Cys226, or from Pro230, to the carboxyl-terminus thereof. TheC-terminal lysine (residue 447 according to the EU numbering system) ofthe Fc region may be removed, for example, during production orpurification of the antibody, or by recombinantly engineering thenucleic acid encoding a heavy chain of the antibody. Accordingly, acomposition of intact antibodies may comprise antibody populations withall K447 residues removed, antibody populations with no K447 residuesremoved, and antibody populations having a mixture of antibodies withand without the K447 residue. Suitable native-sequence Fc regions foruse in the antibodies of the invention include human IgG1, IgG2 (IgG2S,IgG2B), IgG3 and IgG4. “Fc receptor” or “FcR” describes a receptor thatbinds to the Fc region of an antibody. The preferred FcR is a nativesequence human FcR. Moreover, a preferred FcR is one which binds an IgGantibody (a gamma receptor) and includes receptors of the FcγRI, FcγRII,and FcγRIII subclasses, including allelic variants and alternativelyspliced forms of these receptors, FcγRII receptors include FcγRIIA (an“activating receptor”) and FcγRIIB (an “inhibiting receptor”), whichhave similar amino acid sequences that differ primarily in thecytoplasmic domains thereof. Activating receptor FcγRIIA contains animmunoreceptor tyrosine-based activation motif (ITAM) in its cytoplasmicdomain. Inhibiting receptor FcγRIIB contains an immunoreceptertyrosine-based inhibition motif (ITIM) in its cytoplasmic domain, (seeM. Daeron, Annu. Rev. Immunol. 15:203-234 (1997). FcRs are reviewed inRavetch and Kinet, Annu. Rev. Immunol. 9: 457-92 (1991); Capel et al,Immonomethods 4: 25-34 (1994); and de Haas et al, J. Lab. Clin. Med.126: 330-41 (1995). Other FcRs, including those to be identified in thefuture, are encompassed by the term “FcR” herein.

The term “Fc receptor” or “FcR” also includes the neonatal receptor,FcRn, which is responsible for the transfer of maternal IgGs to thefetus. Guyer et al, J. Immunol. 117: 587 (1976) and Kim et al., J.Immunol. 24: 249 (1994). Methods of measuring binding to FcRn are known(see, e.g., Ghetie and Ward, Immunol. Today 18: (12): 592-6 (1997);Ghetie et al, Nature Biotechnology 15 (7): 637-40 (1997); Hinton at al,J. Biol. Chem. TJI (8): 6213-6 (2004); WO 2004/92219 (Hinton et al).Binding to FcRn in vivo and serum half-life of human FcRn high-affinitybinding polypeptides can be assayed, e.g., in transgenic mice ortransfected human cell lines expressing human FcRn, or in primates towhich the polypeptides having a variant Fc region are administered. WO2004/42072 (Peste) describes antibody variants which improved ordiminished binding to FcRs. See also, e.g., Shields et al, J. Biol.Chem. 9(2): 6591-6694 (2001).

“Effector cells” are leukocytes which express one or more FcRs andperform effector functions. In one aspect, the effector cells express atleast FcγRIII and perform ADCC effector function. Examples of humanleukocytes which mediate ADCC include peripheral blood mononuclear cells(PMC), natural killer (NK) cells, monocytes, cytotoxic T cells andneutrophils. The effector cells may be isolated from a native source,e.g., blood. Effector cells generally are lymphocytes associated withthe effector phase, and function to produce cytokines (helper T cells),killing cells in infected with pathogens (cytotoxic T cells) orsecreting antibodies (differentiated B cells).

“Binding affinity” generally refers to the strength of the sum total ofnon-covalent interactions between a single binding site of a molecule(e.g., of an antibody) and its binding partner (e.g., an antigen).Unless indicated otherwise, as used herein, “binding affinity”, “bindto”, “binds to” or “binding to” refers to intrinsic binding affinitythat reflects a 1:1 interaction between members of a binding pair (e.g.,antibody Fab fragment and antigen). The affinity of a molecule X for itspartner Y can generally be represented by the dissociation constant(K_(D)). Affinity can be measured by common methods known in the art,including those described herein. Low-affinity antibodies generally bindantigen slowly and tend to dissociate readily, whereas high-affinityantibodies generally bind antigen faster and tend to remain boundlonger. A variety of methods of measuring binding affinity are known inthe art, any of which can be used for purposes of the present invention.Specific illustrative and exemplary embodiments for measuring bindingaffinity, i.e. binding strength are described in the following.

The “K_(D)” or “K_(D) value” according to this invention is in oneembodiment measured by a radiolabeled antigen binding assay (RIA) perwith the Fab version of the antibody and antigen molecule as describedby the following assay that measures solution binding affinity of Fabsfor antigen by equilibrating Fab with a minimal concentration of(¹²⁵I)-labeled antigen in the presence of a titration series ofunlabeled antigen, then capturing bound antigen with an anti-Fabantibody-coated plate (Chen, et al, (1999) J. Mol Biol 293:865-881). Toestablish conditions for the assay, microtiter plates (Dynex) are coatedovernight with 5 μg/ml of a capturing anti-Fab antibody (Cappel Labs) in50 mM sodium carbonate (pH 9.6), and subsequently blocked with 2% (w/v)bovine serum albumin in PBS for two to five hours at room temperature(approximately 23° C.). In a non-adsorbent plate (Nunc #269620), 100 pMor 26 pM [¹²⁵I]-antigen are mixed with serial dilutions of a Fab ofinterest (consistent with assessment of an anti-VEGF antibody, Fab-12,in Presta et al, (1997) Cancer Res. 57:4593-4599). The Fab of interestis then incubated overnight; however, the incubation may continue for alonger period (e.g., 65 hours) to ensure that equilibrium is reached.Thereafter, the mixtures are transferred to the capture plate forincubation at room temperature for one hour. The solution is thenremoved and the plate washed eight times with 0.1% Tween-20 in PBS. Whenthe plates have dried, 150 μl/well of scintillant (Micro Scint-20;Packard) is added, and the plates are counted on a Topcount gammacounter (Packard) for ten minutes. Concentrations of each Fab that giveless than or equal to 20% of maximal binding are chosen for use incompetitive binding assays. According to another embodiment, the K_(D)is measured by using surface-plasmon resonace essays using aBIACORE®-2000 or a BIACORE®-3000 instrument (BIAcore, Inc., Piscataway,N.J.) at 25° C. with immobilized antigen CM5 chips at −10 response units(RU). Briefly, carboxymethylated dextran biosensor chips (CM5, BIAcoreInc.) are activated with N-ethyl-N′-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) andN-hydroxysuccinimide (NHS) according to the supplier's instructions.Antigen is diluted with 10 mM sodium acetate, pH 4.8, to 5 μg/ml (−0.2μM) before injection at a flow rate of 5 μL/minute to achieveapproximately 10 response units (RU) of coupled protein. Following theinjection of antigen, 1 M ethanolamine is injected to block unreactedgroups. For kinetics measurements, two-fold serial dilutions of Fab(0.78 nM to 500 nM) are in PBS with 0.05% TWEEN 20™ surfactant (PBST) at25° C. at a flow rate of approximately 25 μL/min. Association rates(k_(on)) and dissociation rates (k_(off)) are calculated using a simpleone-to-one Langmuir binding model (BIAcore Evaluation Software version3.2) by simultaneously fitting the association and dissociationsensorgrams. The equilibrium dissociation constant (K_(D)) is calculatedas the ratio k_(off)/k_(on). See, e.g., Chen et al, J. Mol. Biol.293:865-881 (1999). If the on-rate exceeds 10⁶M⁻¹s⁻¹ by thesurface-plasmon resonance assay above, than the on-rate can bedetermined by using a fluorescent quenching technique that measures theincrease or decrease in fluorescence-emission intensity (excitation=295nm, emission=340 nm, 16 nm band-pass) at 25° C. of a 20 nM anti-antigenantibody (Fab form) in PBS, pH 7.2, in the presence of increasingconcentrations of antigen as measured in a spectrometer, such as astop-flow-equipped spectrophotometer (Aviv Instruments) or a 8000-seriesSLM-AMINCO™ spectrophotometer (ThermoSpectronic) with a stirred cuvette.

An “on-rate” “rate of association” “association rate” or “k_(on)”according to this invention can also be determined as described aboveusing a BIACORE®-2000 or a BIACORE®- 3000 system (BIAcore, Inc.,Piscataway, N.J.) at 25° C. with immobilized antigen CM5 chips at about10 response units (RU). Briefly, carboxymethylated dextran biosensorships (CM5, BIAcore Inc.) are activated with N-ethyl-N′-(3-dimethylaminopropyl)-carbodiimide hydrochloride (ECD) and N-hydroxysuccinimide (NHS)according to the supplier's instructions. Antigen is diluted with 10 mMsodium acetate, ph 4.8, into 5 mg/ml (˜0.2 mM) before injection at aflow rate of 5 ml/min. to achieve approximately 10 response units (RU)of coupled protein. Following the infection of antigen, IM ethanolamineis added to block unreacted groups. For kinetics measurements, two-foldserial dilutions of Fab (0.78 nM to 500 nM) are injected in PBS with0.05% Tween 20 (PBST) at 25° C. at a flow rate of approximately 25μl/min. Association rates (k_(on)) and dissociation rates (k_(o)ff) arecalculated using a simple one-to-one Langmuir binding model (BIAcoreEvaluation Software version 3.2) by simultaneous fitting the associationand dissociation sensorgram. The equilibrium dissociation constant(K_(D)) was calculated as the ratio k_(off)/k_(on). See, e.g., Chen, Y.,et al, (1999) J. Mol Biol 293:865-881. However, if the on-rate exceeds10⁶ M⁻¹ s⁻¹ by the surface plasmon resonance assay above, then theon-rate is preferably determined by using a fluorescent quenchingtechnique that measures the increase or decrease in fluorescenceemission intensity (excitation=295 nm; emission=340 nm, 16 nm band-pass)at 25° C. of a 20 nM anti-antigen antibody (Fab form) in PBS, pH 7.2, inthe presence of increasing concentrations of antigen as measured in a aspectrometer, such as a slop-flow equipped spectrophometer (Avivinstruments) or a 8000-series SLM-Aminco spectrophotometer (ThermoSpectronic) with a stirred cuvette.

The term “functional epitope” as used herein refers to amino acidresidues of an antigen that contribute energetically to the binding ofan antibody, i.e. forming an “energetic epitope”. Mutation of any one ofthe energetically contributing residues of the antigen to alanine willdisrupt the binding of the antibody such that the relative K_(D) ratio(K_(D)mutant PD-L1/K_(D) wild type PD-L1) of the antibody will begreater than 4 (see Example 3.x(b)).

The term “conformational epitope” as used herein refers to amino acidresidues of the PD-L1 antigen that come together on the surface when thepolypeptide chain folds to form the native protein, and show asignificantly reduced rate of HD exchange due to Fab binding, asdescribed in the experimental section. The conformation epitopecontains, but is not limited to, the functional epitope.

The phrase “substantially reduced,” or “substantially different,” asused herein, denotes a sufficiently high degree of difference betweentwo numeric values (generally one associated with a molecule and theother associated with a reference/comparator molecule) such that one ofskill in the art would consider the difference between the two values tobe of statistical significance within the context of the biologicalcharacteristic measured by said values (e.g., K_(D) values). Thedifference between said two values is, for example, greater than about10%, greater than about 20%, greater than about 30%, greater than about40%, and/or greater than about 50% as a function of the value for thereference/comparator molecule.

The term “substantially similar” or “substantially the same,” as usedherein, denotes a sufficiently high degree of similarity between twonumeric values (for example, one associated with antibody of theinvention and the other associated with a reference/comparatorantibody), such that one of skill in the art would consider thedifference between the two values to be of little or no biologicaland/or statistical significance within the context of the biologicalcharacteristic measured by said values {e.g., K_(D) values). Thedifference between said two values is, for example, less than about 50%,less than about 40%, less than about 30%, less than about 20%, and/orless than about 10% as a function of the reference/comparator value.

“percent (%) amino acid sequence identity” and “homology” with respectto a peptide, polypeptide or antibody sequence are defined as thepercentage of amino acid residues in a candidate sequence that areidentical with the amino acid residues in the specific peptide orpolypeptide sequence, after aligning the sequences and introducing gaps,if necessary, to achieve the maximum percent sequence identity, and notconsidering any conservative substitutions as part of the sequenceidentity. Alignment for purposes of determining percent amino acidsequence identity can be achieved in various ways that are within theskill in the art, for instance, using publicly available computersoftware such as BLAST, BLAST-2 or ALIGN software. Those skilled in theart can determine appropriate parameters for measuring alignment,including any algorithms needed to achieve maximal alignment over thefull length of the sequences being compared.

A “blocking” antibody or an “antagonist” antibody is one that inhibitsor reduces a biological activity of the antigen it binds to. In someembodiments, blocking antibodies or antagonist antibodies substantial orcompletely inhibit the biological activity of the antigen. Theanti-PD-L1 antibodies of the invention block the interaction betweenPD-L1 and its receptor PD-1, and thus the signaling through PD-1 so asto restore a functional response by T-cells from a dysfunctional stateto antigen stimulation. An “agonist” or activating antibody is one thatenhances or initiates signaling by the antigen to which it binds. Insome embodiments, agonist antibodies cause or activate signaling withoutthe presence of the natural ligand.

The terms “cross-compete”, “cross-competition”, “cross-block”,“cross-blocked” and “cross-blocking” are used interchangeably herein tomean the ability of an antibody or fragment thereof to interfere withthe binding directly or indirectly through allosteric modulation of theanti-PD-L1 antibodies of the invention to the target human PD-L1. Theextent to which an an antibody or fragment thereof is able to interferewith the binding of another to the target, and therefore whether it canbe said to cross-block or cross-complete according to the invention, canbe determined using competition binding assays. One particularlysuitable quantitative cross-competition assay uses a FACS- or anAlphaScreen-based approach to measure competition between the labeled(e.g. His tagged, biotinylated or radioactive labelled) an antibody orfragment thereof and the other an antibody or fragment thereof in termsof their binding to the target. In the Experimental Section a suitableassay is described for determining whether a binding moleculecross-competes or is capable of cross-competing with an antibody orfragment thereof. In general, a cross-competing antibody or fragmentthereof is for example one which will bind to the target in thecross-competition assay such that, during the assay and in the presenceof a second antibody or fragment thereof, the recorded displacement ofthe immunoglobulin single variable domain or polypeptide according tothe invention is up to 100% (e.g. in FACS based competition assay) ofthe maximum theoretical displacement (e.g. displacement by cold (e.g.unlabeled) antibody or fragment thereof that needs to be cross-blocked)by the to be tested potentially cross-blocking antibody or fragmentthereof that is present in given amount. Preferably, cross-competingantibodies or fragments thereof have a recorded displacement that isbetween 10% and 100%, more preferred between 50% to 100%.

An “isolated” nucleic acid molecule encoding the antibodies herein is anucleic acid molecule that is identified and separated from at least onecontaminant nucleic acid molecule with which it is ordinarily associatedin the environment in which it was produced. Preferably, the isolatednucleic acid is free of association with all components associated withthe production environment. The isolated nucleic acid molecules encodingthe polypeptides and antibodies herein is in a form other than in theform or setting in which it is found in nature. Isolated nucleic acidmolecules therefore are distinguished from nucleic acid encoding thepolypeptides and antibodies herein existing naturally in cells.

The term “control sequences” refers to DNA sequences necessary for theexpression of an operably linked coding sequence in a particular hostorganism. The control sequences that are suitable for prokaryotes, forexample, include a promoter, optionally an operator sequence, and aribosome binding site. Eukaryotic cells are known to utilize promoters,polyadenylation signals, and enhancers. Nucleic acid is “operablylinked” when it is placed into a functional relationship with anothernucleic acid sequence. For example, DNA for a presequence or secretoryleader is operably linked to DNA for a polypeptide if it is expressed aspreprotein that participates in the secretion of the polypeptide; apromoter or enhancer is operably linked to a coding sequence if itaffects the transcription of the sequence; or a ribosome binding site isoperably linked to a coding sequence if it is positioned so as tofacilitate translation. Generally “operably linked” means that the DNAsequences being linked are contiguous, and, in the case of a secretoryleader, contiguous and in reading phase. However, enhancers do not haveto be contiguous. Linking is accomplished by ligation at convenientrestriction sites. If such sites do not exist, the syntheticoligonucleotide adaptors or linkers are used in accordance withconventional A “stable” formulation is one in which the protein thereinessentially retains its physical and chemical stability and integrityupon storage. Various analytical techniques for measuring proteinstability are available in the art and are reviewed in Peptide andProtein Drug Delivery, 247-301, Vincent Lee Ed., Marcel Dekker, Inc.,New York, N.Y., Pubs. (1991) and Jones, A. Adv. Drug Delivery Rev.10:29-90 (1993). Stability can be measured at a selected temperature fora selected time period. For rapid screening, the formulation may be keptat 40° C. for 2 weeks to 1 month, at which time stability is measured.Where the formulation is to be stored at 2-8° C., generally theformulation should be stable at 30° C. or 40° C. for at least 1 monthand/or stable at 2-8° C. for at least 2 years. Where the formulation isto be stored at 30° C., generally the formulation should be stable forat least 2 years at 30° C. and/or stable at 40° C. for at least 6months. For example, the extent of aggregation during storage can beused as an indicator of protein stability, Thus, a “stable” formulationmay be one wherein less than about 10% and preferably less than about 5%of the protein are present as an aggregate in the formulation. In otherembodiments, any increase in aggregate formation during storage of theformulation can be determined.

A “reconstituted” formulation is one which has been prepared bydissolving a lyophilized protein or antibody formulation in a diluentsuch that the protein is dispersed throughout. The reconstitutedformulation is suitable for administration (e.g. subscutaneousadministration) to a patient to be treated with the protein of interestand, in certain embodiments of the invention, may be one which issuitable for parenteral or intravenous administration.

An “isotonic” formulation is one which has essentially the same osmoticpressure as human blood. Isotonic formulations will generally have anosmotic pressure from about 250 to 350 mOsm. The term “hypotonic”describes a formulation with an osmotic pressure below that of humanblood. Correspondingly, the term “hypertonic” is used to describe aformulation with an osmotic pressure above that of human blood.Isotonicity can be measured using a vapor pressure or ice-freezing typeosmometer, for example. The formulations of the present invention arehypertonic as a result of the addition of salt and/or buffer. “Carriers”as used herein include pharmaceutically acceptable carriers, excipients,or stabilizers that are nontoxic to the cell or mammal being exposedthereto at the dosages and concentrations employed. Often thephysiologically acceptable carrier is an aqueous pH buffered solution.Examples of physiologically acceptable carriers include buffers such asphosphate, citrate, and other organic acids; antioxidants includingascorbic acid; low molecular weight (less than about 10 residues)polypeptide; proteins, such as serum albumin, gelatin, orimmunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone;amino acids such as glycine, glutamine, asparagine, arginine or lysine;monosaccharides, disaccharides, and other carbohydrates includingglucose, mannose, or dextrins; chelating agents such as EDTA; sugaralcohols such as mannitol or sorbitol; salt-forming counterions such assodium; and/or nonionic surfactants such as TWEEN™, polyethylene glycol(PEG), and PLURONICS™.

A “pharmaceutically acceptable acid” includes inorganic and organicacids which are non toxic at the concentration and manner in which theyare formulated. For example, suitable inorganic acids includehydrochloric, perchloric, hydrobromic, hydroiodic, nitric, sulfuric,sulfonic, sulfuric, sulfanilic, phosphoric, carbonic, etc. Suitableorganic acids include straight and branched-chain alkyl, aromatic,cyclic, cycloaliphatic, arylaliphatic, heterocyclic, saturated,unsaturated, mono, di- and tri-carboxylic, including for example,formic, acetic, 2-hydroxyacetic, trifluoroacetic, phenylacetic,trimethylacetic, t-buyl acetic, anthranilic, propanoic,2-hydroxypropanoic, 2-oxopropanoic, propandioic, cyclopentanepropionic,cyclopentane propionic, 3-phenylpropionic, butanoic, butandioic,benzoic, 3-(4-hydroxybenzoyl)benzoic, 2-acetoxy-benzoic, ascorbic,cinnamic, lauryl sulfuric, stearic, muconic, mandelic, succinic,embonic, fumaric, malic, maleic, hydroxymaleic, malonic, lactic, citric,tartaric, glycolic, glyconic, gluconic, pyruvic, glyoxalic, oxalic,mesylic, succinic, salicylic, phthalic, palmoic, palmeic, thiocyanic,methanesulphonic, ethanesulphonic, 1,2-ethanedisulfonic,2-hydroxyethanesulfonic, benzenesulphonic, 4-chorobenzenesulfonic,napthalene-2-sulphonic, p-toluenesulohonic, camphorsulphonic,4-methylbicyclo[2.2.2]-oct-2-ene-I-carboxylic, glucoheptonic,4,4′-methylenebis-3-(hydroxy-2-ene-I-carboxylic acid), hydroxynapthoic.

“Pharmaceutically-acceptable bases” include inorganic and organic baseswhich are non-toxic at the concentration and manner in which they areformulated. For example, suitable bases include those formed frominorganic base forming metals such as lithium, sodium, potassium,magnesium, calcium, ammonium, iron, zinc, capper, manganese, aluminum,N-methylglucamine, morpholine, piperidine and organic nontoxic basesincluding, primary, secondary and tertiary amines, substituted amines,cyclic amines and basic ion exchange resins, [e.g., N(R′)₄ ⁺ (where R′is independently H or C₁₋₄ alkyl, ammonium, Tris)], for example,isopropylamine, trimethylamine, diethylamine triethylamine,tripropylamine, ethanolamine, 2-diethylaminoethanol, trimethaminedicyclohexylamine, lysine, arginine, histidine, caffeine, procaine,hydrabamine, choline, betaine, ethylenediamine, glucosamine,methylglucamine, theobromine, purines, piperazine, piperidine,N-ethylpiperidine, polyamine resins and the like. Particularly preferredorganic non-toxic bases are isopropylamine, diethylamine, ethanolamine,trimethamine, dicyclohexylamine, choline, and caffeine. Additionalpharmaceutically acceptable acids and bases useable with the presentinvention include those which are derived from the amino acids, forexample, histidine, glycine, phenylalanine, aspartic acid, glutamicacid, lysine and asparagine.

“Pharmaceutically acceptable” buffers and salts include those derivedfrom both acid and base addition salts of the above indicated acids andbases. Specific buffers and/or salts include histidine, succinate andacetate.

A “pharmaceutically acceptable sugar” is a molecule which, when combinedwith a protein of interest, significantly prevents or reduces chemicaland/or physical instability of the protein upon storage. When theformulation is intended to be lyophilized and then reconstituted,“pharmaceutically acceptable sugars” may also be known as a“lyoprotectant”. Exemplary sugars and their corresponding sugar alcoholsinclude: an amino acid such as monosodium glutamate or histidine; amethylamine such as betaine; a lyotropic salt such as magnesium sulfate;a polyol such as trihydric or higher molecular weight sugar alcohols,e.g. glycerin, dextran, erythritol, glycerol, arabitol, xylitol,sorbitol, and mannitol; propylene glycol; polyethylene glycol;PLURONICS®, and combinations thereof. Additional exemplarylyoprotectants include glycerin and gelatin, and the sugars mellibiose,melezitose, raffmose, mannotriose and stachyose. Examples of reducingsugars include glucose, maltose, lactose, maltutose, iso-maltulose andlactulose. Examples of non-reducing sugars include non-reducingglycosides of polyhydroxy compounds selected from sugar alcohols andother straight chain polyalcohols. Preferred sugar alcohols aremonoglycosides, especially those compounds obtained by reduction ofdisaccharides such as lactose, maltose, lactulose and maltulose. Theglycosidic side group can be either glucosidic or galactosidic.Additional examples of sugar alcohols are glucitol, maltitol, lactitoland iso-maltulose. The preferred pharmaceutically-acceptable sugars arethe non-reducing sugars trehalose or sucrose. Pharmaceuticallyacceptable sugars are added to the formulation in a “protecting amount”(e.g. pre-lyophilization) which means that the protein essentiallyretains its physical and chemical stability and integrity during storage(e.g., after reconstitution and storage).

The “diluent” of interest herein is one which is pharmaceuticallyacceptable (safe and non-toxic for administration to a human) and isuseful for the preparation of a liquid formulation, such as aformulation reconstituted after lyophilization. Exemplary diluentsinclude sterile water, bacteriostatic water for infection (BWFI), a pHbuffered solution (e.g. phosphate-buffered saline), sterile salinesolution, Ringer's solution or dextrose solution. in an alternativeembodiment, diluents can include aqueous solutions of salts and/orbuffers.

A “preservative” is a compound which can be added to the formulationsherein to reduce bacterial activity. The addition of a preservative may,for example, facilitate the production of a multi-use (multiple-dose)formulation. Examples of potential preservatives includeoctadecyldimethylbenzyl ammonium chloride, hexamethonium chloride,benzalkonium chloride (a mixture of alkylbenzyldimethylammoniumchlorides in which the alkyl groups are long-chain compounds), andbenzethonium chloride. Other types of preservatives include aromaticalcohols such as phenol, butyl and benzyl alcohol, alkyl parabens suchas methyl or propyl paraben, catechol, resorcinol, cyclohexanol,3-pentanol, and w-cresol. The most preferred preservative herein isbenzyl alcohol.

“Treatment” refers to clinical intervention designed to alter thenatural course of the individual or cell being treated, and can beperformed either for prophylaxis or during the course of clinicalpathology. Desirable effects of treatment include preventing occurrenceor recurrence of disease, preventing metastasis, decreasing the rate ofdisease progression, ameliorating or palliating the disease state, andremission or improved prognosis. In some embodiments, antibodies of theinvention are used to delay development of a disease or disorder. Asubject is successfully “treated”, for example, using the apoptotioanti-PD-L1 antibodies of the invention if one or more symptomsassociated with a T-cell dysfunctional disorder is mitigated.

An “effective amount’ refers to at least an amount effective, at dosagesand for periods of time necessary, to achieve the desired or indicatedeffect, including a therapeutic or prophylactic result. For example, aneffective amount of the anti-PD-L1 antibodies of the present inventionis at least the minimum concentration that results in inhibition ofsignaling from PD-L1, either through PD-1 on T-cells or B7.1 on otherAPCs or both.

A “therapeutically effective amount” is at least the minimumconcentrations required to effect a measurable improvement or preventionof a particular disorder. A therapeutically effective amount herein mayvary according to factors such as the disease state, age, sex, andweight of the patient, and the ability of the antibody to elicit adesired response in the individual. A therapeutically effective amountis also one in which any toxic or detrimental effects of the antibodyare outweighed by the therapeutically beneficial effects. For example, atherapeutically effective amount of the anti-PD-L1 antibodies of thepresent invention is at least the minimum concentration that results ininhibition of at least one symptom of a T cell dysfunctional disorder.

A “prophylactically effective amount” refers to an amount effective, atthe dosages and for periods of time necessary, to achieve the desiredprophylactic result. For example, a prophylactically effective amount ofthe ant-PD-L1 antibodies of the present invention is at least theminimum concentration that prevents or attenuates the development of atleast one symptom of a T cell dysfunctional disorder.

“Mammal” for purposes of treatment refers to any animal classified as amammal, including humans, domestic and farm animals, and zoo, sports, orpet animals, such as dogs, horses, rabbits, cattle, pigs, hamsters,gerbils, mice, ferrets, rats, cats, etc. Preferably, the mammal ishuman.

The term “pharmaceutical formulation” refers to a preparation that is insuch form as to permit the biological activity of the active ingredientto be effective, and that contains no additional components that areunacceptably toxic to a subject to which the formulation would beadministered. Such formulations are sterile.

A “sterile” formulation is aseptic or free from all livingmicroorganisms and their spores.

The term “about” as used herein refers to the usual error range for therespective value readily known to the skilled person in this technicalfield.

An “autoimmune disorder” is a disease or disorder arising from anddirected against an individual's own tissues, or organs or aco-segregation or manifestation thereof or suiting condition therefrom.Autoimmune diseases can be an organ-specific disease (i.e., the immuneresponse is specifically directed against an organ system such as theendocrine system, the hematopoietic system, the skin, thecardiopulmonary system, the gastrointestinal and liver systems, therenal system, the thyroid, the ears, the neuromuscular system, thecentral nervous system, etc.) or a systemic disease that can affectmultiple organ systems (far example, systemic lupus erythematosus (SLE),rheumatoid arthritis (RA), polymyositis, etc.). Preferred such diseasesinclude autoimmune rheumatologic disorders (such as, for example, RA,Sjogren's syndrome, sclerodemia, lupus such as SLE and lupus nephritis,polymyositis-dermatomyositis, cryoglobulinemia, anti-phosoholipidantibody syndrome, and psoriatic arthritis), autoimmune gastrointestinaland liver disorders (such as, for example, inflammatory bowel diseases{e.g., ulcerative colitis and Crohn's disease), autoimmune gastritis andpernicious anemia, autoimmune hepatitis, primary biliary cirrhosis,primary sclerosing cholangitis, and celiac disease), vasculitis (suchas, for example, ANCA-negative vasculitis and ANCA-associatedvasculitis, including Churg-Strauss vasculitis. Wegener'sgranulomatosis, and microscopic polyangiitis), autoimmune neurologicaldisorders (such as, for example, multiple sclerosis, opsoclonusmyoclonus syndrome, myasthenia gravis, neuromyelitis optica, Parkinson'sdisease, Alzheimer's disease, and autoimmune polyneuropathies), renaldisorders (such as, for example, glomerulonephritis, Goodpasture'ssyndrome, and Berger's disease), autoimmune dermatologic disorders (suchas, for example, psoriasis, urticaria, hives, pemphigus vulgaris,bullous pemphigoid, and cutaneous lupus erythematosus), hematologicdisorders (such as, for example, thrombocytopenic purpura, thromboticthrombocytopenic purpura, post-transfusion purpura, and autoimmunehemolytic anemia), atherosclerosis, uvetis, autoimmune hearing diseases(such as, for example, inner ear disease and hearing loss), Behcet'sdisease, Raynaud's syndrome, organ transplant, and autoimmune endocrinedisorders (such as, for example, diabetic-related autoimmune diseasessuch as insulin-dependent diabetes mellitus (IDDM), Addison's disease,and autoimmune thyroid disease (e.g., Graves' disease and thyroiditis)).More preferred such diseases include, for example, RA, ulcerativecolitis, ANCA-associated vasculitis, lupus, multiple sclerosis,Sjogren's syndrome, Graves' disease, IDDM, pernicious anemia,thyroiditis, and glomerulonephritis.

The term “cytotoxic agent” as used herein refers to a substance thatinhibits or prevents the function of cells and/or causes destruction ofcells. The term includes radioactive isotopes (e.g. At²¹¹, I¹³¹, I¹²⁵,Y⁹⁰, Re¹⁸⁶, Re¹⁸⁸, Sm¹⁵³, Bi²¹², P³² and radioactive isotopes of Lu),and toxins such as small-molecule toxins or ezymatically active toxinsof bacterial, fungal, plant or, animal origin, or fragments thereof.

A “chemotherapeutic agent” is a chemical compound useful in thetreatment of cancer. Examples of chemotherapeutic agents includealkylating agents such as thiotepa and cyclophosphamide; alkylsulfonates such as busulfan, improsulfan, and piposulfan; aziridinessuch as benzodopa, carboquone, meturedopa, and uredopa; ethyleniminesand methylamelamines including altretamine, triethylenemelamine,trietylenephosphoramide, triethiylenethiophosphoramide andtrimethylolomelamine; acetogenins (especially bullatacin andbullatacinone); delta-9-tetrahydrocannabinol (dronabinol):beta-lapachone; lspachol; colchicines; betulinic acid; a camptothecin(including the synthetic analogue topotecan (CPT-11 (irinotecan),acetylcamptothecin, scopolectin, and 9-aminocamptothecin); bryostatin;pemetrexed; callystatin; CC-1065 (including its adozelesin, carzelesinand bizelesin synthetic analogues); podophyllotoxin; podophyllinic acid;teniposide: cryptophycins (particularly cryptophycin 1 and cryptophycin8); dolastatin; duocatmycin (including the synthetic analogues, KW-2189and CB1-TM1); eleutherobin; pancratistatin; TLK-286; CDP323, an oralalpha-4 integrin inhibitor; a sarcodictyin; spongistatin; nitrogenmustards such as chlorambucil, chlomaphazine, cholophosphamide,estramustine, ifosfamide, mechlorethamine, mechlorethamine oxidehydrochloride, melphalan, novembichin, phenesterine, prednimustine,trofostamide, uracil mustard; nitrosureas such as carmustine,chlorozotocin, fotemustine, lomustine, nimustine, and ranimnustine;antibiotics such as the enediyne antibiotics (e. g., calcheamicin,especially calicheamicin gammall and calicheamicin omegall (see, e.g.,Nicolaou et ah, Angew. Chem Intl. Ed. Engl., 33: 183-186 (1994));dynemicin, including dynemicin A; an esperamicin; as well asneocarzinostatin chromophore and related chromoprotein enediyneantibiotic chromophores), aclacinomysins, actinomycin, authramycin,azaserine, bleomycins, cactinomycin, carabicin, carminomycin,carzinophilin, chromomycinis, dactinomycin, daunorubicin, detombicin,6-diazo-5-oxo-L-norleucine, doxombicin (includingmorpholino-doxorubicin, cyanomorpholino-doxorubicin,2-pyrrolino-doxorubicin, doxorubicin HCl liposome injection anddeoxydoxorobicin), epirubicin, esorubicin, idarubicin, marcellomycin,mitomycins such as mitomycin C, mycophenolic acid, nogalamycin,olivomycins, peplomycin, cottromycin, puromycin, quelamycin,rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex,zinostatin, zonibicin; anti-metabolites such as methotrexate,gemcitabine, tegafur, capecitabine, an epothilone, and 5-fluorouracil(5-FU); folic acid analogues such as denopterin, methotrexate,pteropterin, trimetrexate; purine analogs such as fludarabine,6-merceptopurine, thiamiprine, thioguanine; pyrimidine analogs such asancitabine, azacitidine, 6-azauridine, crmofur, cytarabine,dideoxyuridine, doxifluridine, enocitabine, floxuridine, and imatinib (a2-phenylaminopyrimidine derivative), as well as other c-Kit inhibitors;anti-adrenals such as aminoglutethimide, mitotane, trilostane; folicacid replenisher such as frolinic acid; aceglatone; aldophosphamideglycoside; aminolevulinic acid; eniluracil; amsacrine; bestrabucil;bisantrene; edatraxate; defofamine; demecolcine; diaziquone;elfomithine; elliptinium acetate; etoglucid; gallium nitrate;hydroxyurea; lentinan; lonidainine; maytansinoids such as maytansine andansamitocins; mitoguazone; mitoxantrone; mopidanmol; nitraerine;pentostatin; phenamet; pirarubicin; losoxantrone; 2-ethylhydrazide;procarbazine: PSK® polysaccharide complex (JHS Natural Products, EugeneOreg.); razoxane; rhizoxin; sizofiran: spirogermanium; tenuazenic acid;triaziquone; 2,2′,2″-trichlorotriethylamine; trichothecenes (especiallyT-2 toxin, verracurin A, roridin A and anguidine); urethan; vindesine;dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman;gacytosine; arabinoside (“Ara-C”); thiotepa; taxoids, paclitaxel,albumin-engineered nano article formulation of paclitaxel, anddoxetaxel; chloranbucil; 6-thioguanine; mercaptopurine; methotrexate;platinum analogs such as cisplatin and carboplatin; vinblastine;platinum; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine;oxaliplatin; leucovovin; vinorelbine; novantrone; edatrexate;daunomycin; aminopterin; ibandronate; topoisomerase inhibitor RFS 2000;difluoromethylomithine (DMFO); retinoids such as retinoic acid;pharmaceutically acceptable salts, acids or derivatives of any of theabove; as well as combinations of two or more of the above such as CHOP,an abbreviation for a combined therapy of cyclophosphamide, doxorubicin,vinoristine, and prednisolone, and FOLFOX, an abbreviation for atreatment regimen with oxaliplatin combined with 5-FU and leucovovin.

Other therapeutic agents that may be used in combination with theanti-PD-L1 antibodies of the invention are bisphosphonates such asclodronate, NE-58095, zoledronic acid/zoledronate, alendronate,pamidronate, tiludronate, or risodronate; as well as troxacitabine (a1,3-dioxolane nucleoside cytosine analog); anti-sense oligonucleotides,particularly those that inhibit expression of genes in signalingpathways implicated in aberrant cell proliferation, such as, forexample, PKC-alpha, Raf, H-Ras, and epidermal growth factor receptor(EGF-R); vaccines such as Stimuvax vaccine, Theratope vaccine and genetherapy vaccines, for example, Allovectin vaccine, Leuvectin vaccine,and Vaxid vaccine; topoisomerase 1 inhibitor; an anti-estrogen such asfulvestrant; a Kit inhibitor such as imatinib or EXEL-0862 (a tyrosinekinase inhibitor); EGFR inhibitor such as erlotinib or cetuximab; ananti-VEGF inhibitor such as bevacizumab; arinotecan; rmRH; lanatinib andlapatinib ditosylate (an ErbB-2 and EGFR dual tyrosine kinasesmall-molecule inhibitor also known as GW572016); 17AAG (geldanamycinderivative that is a heat shock protein (Hsp) 90 poison), andpharmaceutically acceptable salts, acids or derivatives of any of theabove.

“Stimuvax” is a BLP25 liposome cancer vaccine designed to induce animmune response to cancer cells that express MUC1, a protein antigenwidely expressed on common cancers. MUC1 is over expressed on manycancers such as lung cancer, breast cancer, prostate cancer andcolorectal cancer. Stimuvax is thought to work by stimulating the body'simmune system to identify and destroy cancer cells expressing MUC1,

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows that A09-246-2 efficiently blocks ¹²⁵I-PD-L1 binding toimmobilized PD-1-Fc. Inactive mutant: Mutant VL-A31G,D52E,R99Y ofA09-188-1; A09-246-2 (1): Expressed in HEK 293 cells. A09-246-2 (2):Expressed in CHO-S cells, batch #1. A09-246-2 (3): Expressed in CHO-Scells, batch #2.

FIG. 2 shows sequence of the extracellular domain (fused to a 6 aminoacid His tag, SEQ ID NO:29) of PD-L1. Peptides that could be identifiedby MS are indicated by grey bars. Those that showed protection from HDexchange in the presence of Fab are represented by black bars. Peptidesthat could not be analyzed are highlighted by and italicizing in thesequence.

FIG. 3 shows the epitope of A09-246-2 on PD-L1. The backbone of PD-L1 isshown in a ribbon representation. Amino acids which, when mutated toalanine, destabilize the A09-246-2 PB-L1 binding by more than 0.7kcal/mol are shown as sticks.

FIG. 4 shows that A09-246-2 efficiently enhances T cell activitiesrepresented by IL-2 production as shown by SEA human PBMC assay.

FIGS. 5-18 show that A09-246-2 increases ADCC in different tumor lines(stimulated and non-stimulated) and allotypes.

EXPERIMENTAL SECTION

The working examples presented below are intended to illustrateparticular embodiments of the invention, and are not intended to limitthe scope of the specification or the claims in any way.

1. Selection and Improvement of Antibodies

Antibodies were selected from phage Fab display libraries. The sectionincluded two different arms one utilizing biotinylated human PD-L1 onthe different selection rounds and other alternating human and mousePD-L1 as target on different rounds. 3840 clones were screened by ELISAto identify 170 individual PD-L1 binders. Based on the inhibition ofPD-L1 ligand binding, 48 hits were selected and were expressed in mediumscale for further characterization.

The selected hits were reformatted and expressed as IgGs. Hitoptimization candidates were selected based on the potency to blockbinding of PD-1 to PD-L1 and the ability of binding to both human andmouse versions of PD-L1. Binding to PD-L1 was originally determined byELISA and later quantified by Biacore and binding to PD-L1 expressingcells by FACS. Four candidates fitted the predefined profile, includingA09-188-1 which contained a lambda light chain. A09-188-1 was chosen foraffinity maturation and sequence optimization. The goals of the affinitymaturation were increased affinity to the human target, cross-reactivityto the murine target, and improvement of manufacturability. Heavy chainmutations in the HVR's were introduced by codon based randomization.This heavy chain diversity was combined with light chain diversityintroduced by light chain shuffling to generate the affinity maturationlibrary. Further heavy and light chain FR and HVR residues were mutatedto increase stability of the antibody and introduce amino acids found inthe germline, such as the heavy chain FR mutation I93V.

This yielded the HVR sequences given below. It could be shown that atleast the residues X₁-X₁₇ are variable in terms of target binding andhave preferred meanings as herein disclosed.

-   the HVR-H1 sequence is X₁YX₂MX₃ (SEQ ID NO:1);-   the HVR-H2 sequence is SIYPSGGX₄TFYADX₅VKG (SEQ ID NO:2);-   the HVR-H3 sequence is IKLGTVTTVX₆Y (SEQ ID NO:3);-   wherein: X₁ is K, R, T, Q, G, A, W, M, I or S; X₂ is V, R, K, L, M    or I; X₃ is H, T, N, Q, A, V, Y, W, F or M; X₄ is F or I; X₅ is S or    T; X₆ is E or D.-   HVR-L1 sequence is TGTX₇X₈DVGX₉YNYVS (SEQ ID NO:8);-   HVR-L2 sequence is X₁₀VX₁₁X₁₂RPS (SEQ ID NO:9);-   HVR-L3 sequence is SSX¹³TX₁₄X₁₅X₁₆X₁₇RV (SEQ ID NO:10);-   wherein: X₇ is N or S; X₈ is T, R or S; X₉ is A or G; X₁₀ is E or D;    X₁₁ is I, N or S; X₁₂ is D, H or N; X₁₃ is F or Y; X₁₄ is N or S;    X₁₅ is R, T or S; X₁₆ is G or S; X₁₇ is I or T.

2. Manufacturing, Purification and Formulation 2.1 Bioproduction andClarification

Antibody A09-246-2 corresponding to SEQ ID NO:32 (heavy chain) and SEQID NO:33 (light chain), was expressed from CHO-S cells transfected withthe KOL isotype DNA sequence and sequence-optimized EU version,respectively. Cell cultures were conducted in batch mode in a 250LSingle-use-Bioreactor (SUB) (Table 2-2). Cells were grown in ProCHO5growth media supplemented with 4 mM L-Glutamine±25 μg/mL puromycin at37° C. The cultures were fed with 15% Efficient Feed B and 1.0 mMvalproic acid 3 days after inoculation.

Crude conditioned media from the bioreactor runs were clarified using1.1 m2 Millistak+Pod D0HC (Millipore MD0HC10FS1) and 0.11 m2Millistak+Pod A1HC (Millipore MA1 HC01FS1) filters, followed by terminalfiltration with a Sartopore 2 filter (Sartorius 5445307H8-SS).

2.2 Purification

The purification process consisted of two chromatography steps; (a)MabSelect Protein A to capture the antibody from the clarified harvest,and (b) Hydroxyapatite Type II polish step to remove remainingaggregated product, host cell proteins and DNA, and product relatedimpurities. An intermediate Q-filtration step was inserted between the 2chromatography steps to further reduce DNA. SDS-PAGE and size exclusionchromatography SE-HPLC were used to analyze in-process samples duringpurification. Protein content of the Mabselect in-process samples wasperformed using the Protein A HPLC method while UV/Vis spectroscopy wasused for all other process steps.

Post Mabselect eluates were subjected to 30 min of low pH viralinactivation (pH 3.7) and subsequently neutralized to pH 7.0 prior tothe next purification step.

The final polishing step was the hydroxyapatite Type II Chromatography.The conductivity of the Sartobind Q filtrate was adjusted to <3 mS/cmwith water, and pH reduced to 6.5 with acetic acid before sampleloading.

Bound anti-PD-L1 product was eluted with a NaCl step gradient.Aggregated product-related impurities was eluted with the Strip Buffer.

2.3 Formulation, Ultrafiltration and Diafiltration

Purified anti-PDL1 from the hydroxyapatite polishing step wereconcentrated and then diafiltered into their respective buffersaccording to the Table below. The bulk products were thensterile-filtered through 0.2 μ filter units and further diluted withformulation buffer to their final concentrations. Formulated bulksubstance were further tested for endotoxin and checked by SE-HPLC.

Formulation UF/DF A09-246-2 Starting Sample (mg) 1279 % Recovery 100Final Concentration (mg/ml) 10.2 Purity (% Monomer) 99 FormulationBuffer 10 mM sodium acetate, 140 mM sodium chloride, 0.05% (v/v) Tween20, pH 6.0

2.4 Human Formulation

The following target administration and formulation profile was set:

-   Route of administration: iv infusion-   Human dose range: 1-15 mg/kg-   Concentration: 10 mg/ml-   Storage conditions: liquid or frozen-   Shelf life: more than 12 m

The following liquid formulation was selected:

-   10.0 mg/ml A09-246-2-   10 mM Acetate-   5.1% (w/v) Mannitol-   1.4 mM Methionine-   0.05% (w/v) Tween 20-   adjusted to pH 5.5

The formulation contains antioxidative excipients and was shown to besufficiently stable at the following stress conditions:

-   -   Light stress    -   Shear stress    -   Freeze-thaw cycles    -   Oxidation stress

Stability was assessed at 2-8° C. and 25° C. up to 26 and 13 weeks,respectively. The formulation was found to be sufficiently stable at2-8° C. up to the latest timepoint of 26 weeks. Also, a freeze-driedformulation was made with excellent stability at 25° C. up to 26 weeks.

3. Biochemical and Biological Characterization 3.1 Biacore BindingAffinity and Specificity

Binding affinity and selectivity was determined by Biacore assays. Theaffinity of the lead antibody candidate for human and non humanorthologues is summarized in the table below. The binding affinity ofanti PD-L1 antibody A09-246-2 according to this invention for human,mouse and cynomolgus monkey proteins was statistical similar but highlyreduced for dog, rat and rabbit proteins that displayed a very fastdissociation profile.

PD-L1 ka (1/M s) kd (1/s) KD(M) KD(nM) +/−STDEV Human 2.72E+05 1.83E−046.73E−10 0.7 0.09 Monkey 2.49E+05 2.79E−04 1.12E−09 1.1 0.02 Mouse1.77E+05 1.64E−04 9.26E−10 0.9 0.04 Dog 2.38E+06 1.07E−02 4.50E−09 4.50.4 Rat 3.54E+05 2.20E−02 6.68E−08 66.8 8.8 Rabbit 2.77E+05 2.82E−021.05E−07 105.4 11.2

The kinetic profiles for A09-188-1 and further mutants thereof are shownin the table below:

K_(D) t½ Relative Acc ID anti-PD-L1 antibody (nM) (min) K_(D) A09-168-1Antibody having a heavy chain 5.29 13.2 1.00 according to SEQ ID NO: 34,and a light chain according to SEQ ID NO: 35 Heavy chain combinationvariants of A09-168-1 * A09-204-1 VH-M31I, M33I, M35F, S63T, I93V 0.10576.2 0.02 A09-211-1 VH-M31I, M33L, M35F, S63T, I93V 0.59 109.4 0.11A09-212-1 VH-M33I, M35F, S63T, I93V 0.22 254.4 0.04 A09-213-1 VH-M31I,M35F, S63T, I93V 2.51 27.7 0.47 A09-214-1 VH-M31I, M33I, S63T, I93V 0.40179.1 0.08 A09-215-1 VH-M33L, M35F, S63T, I93V 1.28 50.6 0.24 A09-216-1VH-M31I, M33L, S63T, I93V 0.91 77.8 0.17 A09-219-1 VH-M31S, M33I, M35F,S63T, I93V 0.18 278.5 0.03 A09-220-1 VH-M31S, M33L, M35F, S63T, 0.7868.3 0.15 I93V A09-220-1 VH-M31S, M33I, S63T, I93V 0.44 126.7 0.08A09-222-1 VH-M31S, M33L, S63T, I93V 1.24 47.1 0.23 A09-223-1 VH-M31S,M35F, S63T, I93V 3.62 13.5 0.68 Light chain variant of A09-188-1 *A09-202-1 VL-A31G 4.15 18.8 0.78 Heavy and light chain combinationvariants of A09-188-1 * A09-248-2 VL-A31G, VH- 0.10 436.4 0.02 M31I,M33I, M35F, S63T, I93V A09-239-2 VL-A31G; VH- 0.36 119.7 0.08 M31I,M33L, M35F, S63T, I93V A09-240-2 VL-A31G; VH- 0.16 245.9 0.03 M33I,M33F, S63T, I93V A09-241-2 VL-A31G; VH- 0.32 166.4 0.07 M31I, M33I,S63T, I93V A09-242-2 VL-A31G; VH- 0.76 55.6 0.16 M33L, M35F, S63T, I93VA09-243-2 VL-A31G; VH- 0.63 65.0 0.13 M31I, M33L, S63T, I93V A09-244-2VL-A31G; VH- 0.12 279.7 0.03 M31S, M33I, M35F, S63T, I93V A09-245-2VL-A31G; VH- 0.43 77.2 0.09 M31S, M33L, M35F, S63T, I93V A09-246-2VL-A31G; VH- 0.34 125.4 0.07 M31S, M33I, S63T, I93V A09-247-2 VL-A31G;VH- 0.76 57.8 0.16 M31S, M33L, S63T, I93V * Amino acid positions countedfrom the N-terminus of the heavy and light chains, respectively

3.2 Selectivity

Selectivity was determined by evaluating the binding to members of theB7 family including hu-PD-L1-huFc, hu-PDL-2-huFc, hu-B7.1-huFc,hu-B7.2-huFc, huB7-H2-huFc and huB7-H3-huFc by Biacore.

All the anti-huPD-L1 MAb tested including A09-246-2 reacted specificallywith only huPD-L1 protein and not with any other B7 Family proteins.

3.3 PD-L1: PD-1 Interaction Blocking

The ability of A09-246-2 and a control antibody to compete with thebinding of radio-labelled PD-L1 to immobilized PD-1 was determined byradioactive competitive displacement assay. FIG. 1 shows representativecompetition curves for the test antibodies. The results demonstratedthat A09-246-2 efficiently blocks the interaction of PD-1 and PD-L1 withan IC50 of 0.071±0.008 nM (0.01±0.001 μg/ml).

The follow assay protocol was used:

-   1. Add 60 ml/well of PBS, containing 1 mg/ml a human PD-1Fc (R&D    Systems, 1086-PD; lyophilized PD-1 dissolved with PBS at 200 mg/ml)    to white Costar plates (Corning 3922). Incubate overnight at 4° C.-   2. Rinse wells 1 time with PBS.-   3. Block wells with 120 ml of 0.5% BSA (Sigma A-3059) dissolved in    binding buffer, for 1 h at room to (RT).-   4. Rinse wells 1× with binding buffer.-   5. Add 50 ml of test sample to wells (antibody, supernatant). Dilute    antibodies to 20 nM in assay buffer and serial dilute 9× at a 1:4    dilution. Samples are diluted to 2× final concentration, prior to    adding to the well (usually starting at 10 nM—1× concentration).-   6. Nonspecific binding: add 50 ml of PD-L1/Fc (R&D Systems, 156-B7)    at a final concentration of 250 nM in place of the test sample at a    500 fold excess to the labeled PD-L1. Total wells receive the same    volume of assay buffer.-   7. Add 50 ml of 0.5 nM ¹²⁵I-PD-L1 (custom labeled at Perkin Elmer,    lot number CIS32211, 250 nM, 2400 Ci/mmol) to each well. Dilute to    2× the final concentration in assay buffer—final concentration=0.25    nM.-   8. Shake the plate for 2-2.5 h at 37° C.-   9. Wash the wells 5 times with cold binding buffer.-   10. Add 100 ml of Microscint 20 (Packard 6013641) to each well.    Incubate for at least one h at RT.-   11. Count luminescence on Topcount (¹²⁵I-Microscint protocol).

Binding Buffer: 50 mM Hepes, pH 7.5, 130 mM, NaCl, 5.1 mM KCl, 1.3 mMMgSO₄

Assay buffer: binding buffer+0.5% BSA

3.4 PD-L1: B7.1 Interaction Blocking

The ability of A09-246-2 to block soluble B7.1 binding to PD-L1 on cellsurface was measured by FACS. Results indicated A09-246-2 efficientlyblocks the interaction of B7.1 and PD-L1 with an IC50 of 0.2±0.004 nM(0.03±0.0006 μg/ml).

3.5 Epitope Mapping a) Hydrogen-Deuterium Exchange

The extracellular domain of PD-L1 antigen (SEQ ID NO:29) was incubatedin heavy water (D₂O) solution to allow amide protons on the proteinbackbone to exchange with deuterons from the solvent, in either thepresence or absence of excess anti-PD-L1 Fab or a non-specific Fab. Thesamples were digested with protease and analysed by liquidchromatography-mass spectrometry (LC-MS) to determine the level ofdeuteration in each peptide.

The Fab corresponding to A09-246-2 was used instead of the full IgG inorder to simplify the mass spectrometry analysis by decreasing thenumber of peptides generated by protease digestion. Despite this, someregions remained that could not be identified and analyzed (underlined,italicized sequence portions in FIG. 2), however these regions'representa small fraction of the sequence, and mostly reside in the secondimmunoglobulin domain, distant from the epitope containing region.Residues 32-39 in domain I of the extracellular domain were alsoresistant to identification by mass spectrometry and encompass the siteof an N-linked glycosylation; as A09-246-2 is known to bind anaglycosylated version of PD-L1 produced in E coli, the inability toanalyze this peptide for HD exchange rates was not of concern.

Several peptides from antigen were observed to have a significantlyreduced rate of exchange of protons for deuterons in the presence of Fabthan in its absence, suggesting that at least some residues from thesepeptides are in direct contact with the Fab and constitute aconformational epitope (FIG. 2). Although the two peptides showingprotection from solvent are far apart in the primary sequence(underlined, bold print in FIG. 2, they are proximal in thethree-dimensional structure of PU-L1 and constitute each a singlebinding patch on the surface of the antigen (see FIG. 3).

In summary, HD exchange identified two peptides

-   -   (i) residues 3648 in FIG. 2 (extracellular domain plus His tag,        SEQ ID NO:29), corresponding to residues 54-66 of the full        length sequence (SEQ ID NO:28)    -   (ii) residues 94-104 in FIG. 2 (SEQ ID NO:29), corresponding to        residues 112-122 SEQ ID NO:28        that form a conformational epitope on PD-L1 end that contain the        functional epitope of A09-246-2.

b) Mutegenesis

To obtain a finer, residue-level mapping of the epitope and tocomplement the HD exchange data, molecular modelling and manualinspection of the crystal structure of PD-L1 (Lin, D.Y.-W. et al. PNAS105, 3011-6 (2008; PDB record 3BIK) was used to select solvent exposedresidues within and around the epitope identified by HD exchange. Theselected residues were mutated either to alanine (large to small) or toanother, potentially more disruptive amino acid (small to large).

In total, 48 point mutants were designed, expressed and purified fromHEK cells, and tested for binding to A09-246-2 using surface plasmonresonance (SPR). Binding hotspots, or residues that contribute most tothe binding energy (Wells, J. A., PNAS 93, 1-6, 1996), were identifiedas those that did not meet a threshold binding signal at 100 nM antigen.Furthermore, the affinity of the antibody for wild-type and each mutantwas determined and used to calculate the contribution of each epitoperesidue to the binding energy.

The results are summarized in the table below, where 48 point mutants ofPD-L1 were compared to wild-type PD-L1 antigen for antibody binding. SPR(Biacore) was used to perform a kinetic study allowing determination ofkinetic rate constants (k_(a) and k_(d)). Briefly, goat polyclonalanti-human Fc antibody was chemically coupled to a CM5 chip. A11-128 wasinjected next and captured by the polyclonal. Buffer was used to washout unbound antibody until the baseline RU stabilized. Antigen(wild-type or mutant PD-L1) was next injected at a fixed concentrationfor 3 minutes and the association was recorded. Buffer was injected fora further 3 minutes and dissociation was observed. The antigens wereinjected at concentrations of 100 nM, 50 nM, 25 nM, 12.5 nM and 6.25 nM(except for Y55 and D61 mutants, which were injected at 1 uM, 500 nM,250 nM, 125 nM and 62.5 nM). Between each cycle, the chip wasregenerated with low pH buffer and fresh A09-246-2 was captured prior toinjecting the next concentration of antigen. The rate constants weredetermined by iterative fitting of the data to a 1:1 binding model by analgorithm that minimizes Chi-squared. The equilibrium dissociationconstant (K_(D)) was calculated as the ratio of the kinetic constantsand the change in the Gibbs free energy of binding of mutant relative towild-type PD-L1 (ΔΔG_(mut)) was derived from the ratio of the wild-typeand mutant K_(D)s. The free energy changes are highlighted according todestabilization of antibody-antigen binding; “**”; >3 kcal/moldestabilization (binding hotspots); “*”; >0.7 kcal/mol. Mutants at Y56had such a low affinity that the K_(D) could be accurately measured andthe minimum K_(D) is given instead. For D61A no binding could be found.According to this analysis, amino acids marked with “**” or “*” are partof the functional epitope. The temperature midpoint of fluorescentlymonitored thermal denaturation is given for the wild type and mutantproteins. ND: Not Determined; BP: Biphasic. The qualitative appearanceof the wild type and mutant proteins on size exclusion chromatography(SEC) is also given. M: monodisperse and the same elution volume as wildtype; M/T: peak at the same elution volume as wild type but with anadditional tail. For K_(D) and T_(1/2), the mean and standard deviationis given where n>1.

ΔΔG_(mut) Mutation (kcal/mol) K_(D) (nM) T_(1/2) (° C.) SEC PDL-1  0.000.55 +/− 0.21 59.1 M T20A −0.19 0.39 +/− 0.15 52.5 +/− 0.2 M D26A −0.440.26 +/− 0.19 52.8 +/− 0.2 M L27A −0.07 0.48 +/− 0.68 51.7 +/− 0.5 ME45A −0.54 0.22 58.0 M K46A −0.28 0.34 +/− 0.10 51.6 M Q47A  0.04 0.59+/− 0.27 ND M D49A −0.25 0.36 +/− 0.04 BP (>49) M A51Q  0.09 0.63 +/−0.32 57.3 +/− 0.6 M A52R −0.84 0.13 +/− 0.04 55.2 M I54A −1.28 0.06 +/−0.09 57.2 +/− 2.5 M I54K  0.62 1.57 +/− 0.19 57.2 M Y56A >4**  >1 uM57.5 +/− 0.7 M Y56K >5**  >4 uM 55.4 +/− 1.3 M E58A  1.90* 13.58 +/−0.59  54.6 +/− 0.6 M E60A  1.45* 6.32 +/− 0.44 50.4 M D61A infinite** >5uM 52.0 M K62A  0.49 1.26 +/− 0.07 ND ND N63A  0.21 0.78 +/− 0.18 ND MQ66A  0.86* 2.35 +/− 0.23 ND M V68A  0.02 0.57 +/− 0.04 ND M V68R  0.551.37 +/− 0.05 56.0 M/T H69Q  0.01 0.56 +/− 0.06 ND M E71A −0.25 0.36 +/−0.11 52.8 +/− 1.5 M D73A −0.14 0.43 +/− 0.01 53.5 +/− 2.1 M K75A −0.570.21 +/− 0.06 57.7 +/− 1.8 M V76A −0.49 0.24 +/− 0.06 55.7 M H76A  0.100.65 +/− 0.01 56.6 +/− 0.6 M S79A −0.03 0.52 +/− 0.21 56.3 +/− 0.9 MS79E −0.36 0.30 +/− 0.09 60.0 M S80A  0.07 0.61 +/− 0.05 57.0 M S80E 0.16 0.71 +/− 0.15 56.8 +/− 4.5 M R82A −0.23 0.37 +/− 0.16 51.2 +/− 0.4M K105A −0.19 0.40 +/− 0.08 57.0 +/− 1.5 M Q107A −0.13 0.44 +/− 0.0358.6 +/− 2.0 M/T A109E −0.03 0.52 +/− 0.04 54.0 M V111A −0.42 0.27 +/−0.03 50.2 +/− 0.2 M V111E −0.39 0.28 +/− 0.07 51.6 +/− 0.0 M R113A 1.53* 7.22 +/− 0.26 56.7 M M115A  0.97* 2.79 +/− 0.17 51.4 +/− 0.1 MS117A −0.60 0.20 +/− 0.04 52.7 +/− 0.3 M A121R  0.10 0.46 +/− 0.20 54.0+/− 0.5 M D122A −0.13 0.44 +/− 0.02 ND M Y123A  0.40 1.07 +/− 0.05 ND MK124A  0.10 0.65 +/− 0.09 53.1 +/− 0.6 M R125A  0.41 1.09 +/− 0.04 51.8+/− 0.2 M T127K −0.25 0.36 +/− 0.01 54.0 M T127A −0.13 0.44 +/− 0.0351.4 +/− 0.0 M K129A −0.21 0.38 +/− 0.18 50.8 +/− 1.2 M

It was important to confirm that the lack of binding to A09-246 of theY56A, Y56K and D61A point mutants was indeed due to loss of hotspotresidues and not to global unfolding of the antigen. The structuralintegrity of the mutated proteins was confirmed using a fluorescencemonitored thermal unfolding assay in which the protein is incubated witha dye that is quenched in aqueous solution but fluoresces when bound byexposed hydrophobic residues. As the temperature increases, thermaldenaturation of the protein exposes the hydrophobic core residues andthis can be monitored by an increase in fluorescence of the dye. Mutantsof Y56 or D61 all display a two state transition similar to wild-typePD-L1, indicating a folded structure at room temperature. The data werefit to equation 1 (adapted from Bullock, A. N. et al. Thermodynamicstability of wild-type and mutant p53 core domain. PNAS 94, 14338-14342(1997)) to determine the temperature at the inflection point of thecurve (T_(1/2)).

$\begin{matrix}{F = \frac{\left\{ {{Fi} + {\beta \; i*T} + \left( {\left( {{F\; \max} + {\beta \; \max*T}} \right)*e^{\lbrack{m*{({T - {T\; {1/Z}}})}}\rbrack}} \right)} \right\}}{1 + e^{\lbrack{m*{({T - {T\; {1/2}}})}}\rbrack}}} & {{Equation}\mspace{14mu} 1}\end{matrix}$

Mutants of Y56 and D61 displayed minimal destabilization of the antigenindicated by a small decrease in the T_(1/2) of fluorescence monitoredunfolding (table above). This confirms that Y56 and D61 are true bindinghotspots for A09-246-2. The structural integrity of most of the othermutant proteins was also confirmed by this method (table above). Theobservation that most mutant proteins behaved similarly to wild type onanalytical size exclusion chromatography (last column in the abovetable) provides further support for native structure of mutant antigenproteins.

3.6 Binding to Tumor Cells and Primary Cells

The binding of A09-246-2 to PD-L1 on the surface of tumor cells as wellas on primary human and experimental animal cells was confirmed by aFACS assay. A09-246-2 demonstrated reactivity to human PD-L1 on allseven tested human tumor lines (A431, epithelial carcinoma cell line;A549, lung adenocarcinoma epithelial cell; BxPC3, pancreatic cancercells; HCT116, colorectal carcinoma; M24, melanoma cell lines; PC3mm2,prostate cancer cell line; U-87 MG, glioblastoma-astrocytoma) of whichPD-L1 was up-regulated by interferon treatment to enable detection.Because primary PBMC have low levels of PD-L1 expression which isdifficult to be detected, human PBMC or PBMC from dog, rabbit and ratwere all subjected to PHA stimulation for 2 days. A09-246-2 demonstratedreactivity to PD-L1 on human and animal primary cells.

3.7 EC50 Measured by Direct FACS Binding Assay

The dose dependent binding ability of A09-246-2 to the target on thecell surface was confirmed by FACS. A09-246-2 efficiently binds to humanPD-L1 expressed on the HEK surface with an EC50 of 0.3±0.02 nM(0.04±0.003 μg/ml); to cynomolgus monkey expressed on the HEK cellsurface with an EC50 of 0.94±0.015 nM (0.14±0.002 μg/ml); to mouse PD-L1expressed on the HEK293 cell surface with an EC50 of 0.34±0.08 nM(0.05±0.012 μg/ml) and mouse PD-L1 expressed on the EL4 cell surfacewith an EC50 of 0.91±0.21 nM (0.13±0.03 μg/ml). The assays qualitativelydescribed the dose dependent binding characteristics anti-PD-L1.

3.8 Activity Cellular Assays

Currently there is no scientific evidence that the engagement of PD-L1with it ligands transduces stimulatory signalling through PD-L1 into thePD-L1 expressing cells, therefore the developed assays employed T cellactivation in the procedures. The ability of anti-PD-L1 antibody toenhance T cell immuno-responses was measured in vitro in cellular assaysusing murine T cells or human PBMC.

a) OT-1 Assay

Antigen-specific CD8 T cells were generated by stimulating splenocytesfrom OT-1 transgenic mice with Ova peptide SIINFEKL and cyropreserved.mPD-L1 over-expressing EL4 cells were used as antigen presenting cells.Serial dilutions of tested compounds were incubated with thawed OT-1 Tcells and SIINFEKL-loaded APC for 48 hours. IFN-γ in the supernatant wasmeasured using mIFN-γ ELISA. Anti-PD-L1 (A09-246-2) efficiently enhancedT cell activities represented by IFN-γ production with an EC50 of0.28±0.1 nM (0.04±0.015 μg/ml)

b) SEA Assay

During the human PBMC assay development, it could be demonstrated thatonly anti-PD-L1 treatment did not trigger IL2 or IFN-γ production in theabsence of T cell activation and did not enhance IL-2 production in thepresence of optimal activation either. The ability of anti-PD-L1 toenhance IL-2 production by cells responding to super antigen activationwas assessed. Super antigen such as Staphylococcal enterotoxin A (SEA)is able to crosslink the T cell receptor (TCR) and MHC class II toactivate CD4 cells. The dose dependent activity of A09-246-2 to enhanceT cell functions was assessed upon such activation. Serial dilutions ofA09-246-2 were incubated with human PBMC in the presence of SEA for 96hours. Human IL-2 in the supernatant was measured using human IL-2ELISA. Results indicated anti-PD-L1 efficiently enhanced T cellactivities represented by IL-2 production with an EC50 of 0.08±0.03 mM(0.012±0.005 μg/ml).

3.9 Antibody Dependent Cell-Mediated Cytotoxicity (ADCC)

ADCC was measured utilizing two different human tumor lines A431 andA549 as target cells and human PBMC as effector cells. In some cases,tests were performed using target cells following stimulation withInterferon-gamma to increase the expression of PD-L1. The anti-EGFRantibody, cetuximab, was used as an ADCC positive control. Given thefact that the FcγIIIa receptor 158V allotype displays a higher affinityfor human IgG1 and increases ADCC, the observed results were correlatedwith the donor's allotype.

ADCC activity of A09-246-2 was comparable to that mediated with theanti-EGFR antibody cetuximab, inducing approximately 50% of maximumlysis in both cell lines. INF-γ treatment did not alter the response ofA431 cells for all the different allotypes tested (V/V, V/F and F/F). Asignificant difference (almost twice) between stimulated and notstimulated cells was observed when A549 cells were employed for PBMCfrom V/V and V/F donors. No ADCC was observed when PBMC from F/F donorswere analyzed with A549 cells.

4. In Vivo Activity

In the studies presented here, the efficacy of PD-L1 antibody (Ab)blockade against various murine tumor models was investigated.Inhibition of the PD-L1 interaction is proposed to exert a therapeuticeffect by restoring anti-tumor CD8⁺ T cell responses, thus all of thepreclinical efficacy studies were conducted in syngeneic murine tumormodels in which the immune system of the host is fully intact. Tocircumvent the need for a surrogate antibody, the the antibody used inthe studies was specifically selected for cross-reactivity to murinePD-L1. However, because the antibody is fully human, neutralizingimmunogenicity is elicited in mice, which limits the effective dosingwindow to a seven day period. Despite this significant dosinglimitation, the selected antibody has demonstrated significant activityas a monotherapy and in various combination therapy settings. Theanti-tumor activity of the anti-PD-L1 antibody demonstrated adose-dependent trend when given as a monotherapy against MC38 tumors.

Immunohistochemical analysis of PD-L1 expression within responsive andnon-responsive tumor models revealed a strong link between the level ofPD-L1 expression and the level of anti-tumor efficacy. To confirm theproposed mechanism of action (MOA), a study was conducted in MC38 tumorbearing mice that were systemically depleted of CD8⁺ T cells. In animalsdepleted of CD8⁺ T cells, the efficacy of anti-PD-L1 therapy wascompletely abrogated, confirming that cytotoxic T lymphocyte (CTL)effector function is responsible for the inhibition of tumor growth. Toevaluate the combination potential of anti-PD-L1 therapy, combinationpartners were selected known to elicit anti-tumor cell responses orotherwise enhance the effects of immunotherapy. In combination withfractionated radiotherapy against MC38 tumors, the anti-PD-L1 antibodyshowed strong synergistic activity, with curative potential. Combinationwith a single low-dose of cyclophosphamide resulted in enhancedanti-tumor effects in the MC-38 model that were associated with anincreased frequency of tumor-antigen specific CD8⁺ T cells. Anti-PD-L1therapy significantly extended survival time when combined withGemcitabine in the PANC02 orthotopic tumor model of pancreatic cancer.When anti-PD-L1 was combined with cyclophosphamide pre-treatmentfollowed by vaccination with Stimuvax, a significant increase in tumorgrowth inhibition was achieved in both the MC38/MUC1 and PANC02/MUC1tumor models. Significantly enhanced efficacy was also observed when theanti-PD-L1 antibody was combined with the core components of the FOLFOXchemotherapy regimen. Thus, several promising combination approaches foranti-PD-L1 therapy were successfully identified, including three“standard of care” treatment regimens (radiation therapy; FOLFOX;Gemcitabine).

Mechanistic data derived from these studies demonstrated that anti-PD-L1therapy is consistently associated with increased percentages of CD8⁺ Tcells, CD8⁺ T effector memory mils, and PD-1⁺CD8⁺ T cells in the spleensand tumors of treated mice.

4.1 Dose-Response in MC38 Tumor Model and Combination With CPA

In this study, mice were inoculated subcutaneously in the right flankwith 1×10⁶ MC38 colon carcinoma cells. When tumors reached a mean volumeof ˜50 mm³, mice were sorted into treatment groups (B=14) (defined asstudy day 0). Groups were administered A09-246-2 intravenously at doselevels of 100, 200, 400, or 800 μg days 0, 3, and 6. A control group wastreated with 200 μg of an inactive isotype antibody. Tumors weremeasured twice weekly for the study duration. All treatment groupsdemonstrated significant efficacy (P<0.050) when compared to the isotypecontrol group. Although the 800 μg dose group did not show enhancedefficacy over the 400 μg group, a significant trend toward adose-dependent effect was observed. In a second dose-response study thatfollowed the same design, a general trend toward dose-dependent activitywas again observed. However, the 800 μg dose group in that particularstudy showed significantly lower anti-tumor activity than did the 400 μgdose group. The lack of increased efficacy at doses above 400 μg mayindicate an efficacy plateau as a result of target saturation, or astronger immunogenic effect may occur at higher doses, resulting inlower drug exposure. Additionally, these studies explored the efficacyof anti-PD-L1 in combination with pre-treatment with a low,immunomodulatory dose cyclophosohamide (CPA). The CPA combination wasobserved to significantly improve the efficacy of low doses ofanti-PD-L1 (100 μg), and this effect was associated with increasedfrequencies of o15E tumor antigen-specific CD8⁺ T cells as determined byELISPOT. Immunophenotyping data from these studies revealed thatanti-PD-L1 therapy is associated with significantly increasedpercentages of various CD8⁺ T cell subsets in spleens: total CD8⁺ Tcells, p15E tumor antigen-specific CD8⁺ T cells, PD-1⁺CD8⁺ T cells, andCD8⁺ T effector memory (T_(EM)) and CD8⁺ T central memory (T_(CM))cells. Increased intratumoral accumulation of CD8⁺ T cells and CD8⁺T_(EM) cells was also observed. These observations support thatanti-PD-L1 therapy as an effective strategy for driving anti tumor CD8⁺T cell responses.

4.2 Efficacy in C1498/GFP Disseminated Leukemia Model

To create the disseminated leukemia model, C-4198-GFP leukemia cells(2×10⁴) were injected i.v. into C57BL/6 mice on day 0. Mice were thenrandomized into treatment groups (N=5) that received either a 400 μgdose of anti-PD-L1 Ab (A09-246-2) or an equivalent dose of an inactiveisotype antibody on days 1, 4, and 7 by i.p. injection. The primaryendpoint of this study was survival based on the onset of clinicalsigns, indicative of metastatic dissemination, which warrantedeuthanasia. At the end of the study (day 76), 20% of mice (⅕) were stillalive in the isotype antibody heated group, and 80% (⅘) survivorsremained in the A09-246-2 treated group.

4.3 Combination With Gemcitabine in the PANC02 Orthotopic Model

Three separate studies were conducted to investigate the combination ofthe anti-PD-L1 MAb (A09-246-2) and Gemcitabine (GEM). The studies weredesigned to explore the positioning of anti-PD-L1 therapy within thechemotherapy “holiday” period of a 21 day or 28 day cycle of GEM.Orthotopic models involve the inoculation of tumor cells into the organof origin, resulting in a close recapitulation of disease progression asit occurs in the human setting. To create a model of pancreaticadenocarcinoma, PANC02 cells (1×10⁶) were injected into the pancreas ofC57BL/6 female mice. Five days later, mice were randomized intotreatment groups. GEM was dosed at 150 mg/kg in all studies andA09-246-2 was dosed at 400 μg per mouse. In two studies, a 26 day cycleof GEM was modeled (administration on days 5, 19, 26), with a 14 dayholiday period during which A09-246-2 was given on days 8, 11, 14. In athird study, a 21 day cycle of GEM was modeled (administration on days5, 12, 26, 33), with a 14 day holiday period during which A09-246-2 wasgiven on days 13, 16, 19. Monotherapy with GEM or anti-PD-L1 failed toextend survival time in this model. However, in all three studies, thecombination of GEM and A09-246-2 significantly extended mean survivaltime (P<0.02). Immunophenotyping revealed several effects in groupsreceiving A09-246-2, both as a monotherapy and in combination with GEM,that were consistent with the proposed MOA of anti-PD-L1 includingincreased percentages of CD8⁺ T_(EM) in spleens, an increased ratio ofsplenic CD8⁺ T_(EM) to T_(reg) cells, and increased percentages ofsplenic PD-1⁺CD8⁺ T cells. Furthermore, immunophenotyping of tumorinfiltrating lymphocytes (TIL) showed significantly increasedpercentages of CD8⁺ TIL in the combination group.

4.4 Combination With Low Dose Cyclophosphamide (CPA)

Low-dose CPA is known to enhance anti-tumor immune responses through theinhibition of immunosuppressive regulatory T cells. The potential forlow-dose CPA ore-treatment was investigated to enhance the efficacy ofthe anti-PD-L1 Ab (A09-246-2) in the MC38 subcutaneous tumor model. Micewere inoculated subcutaneously in the right flank with 1×10 ⁶ MC38 coloncarcinoma cells. When tumors reached a mean volume of ˜50 mm³, mice weresorted into treatment groups (N=14) on day 0. The combination groupreceived 100 μg of A09-246-2 by i.v. injection on days 0, 3, and 6, withor without pre-treatment with a 100 mg/kg dose of CPA delivered i.v. onday −1. A control treatment group received 100 μg of an inactive isotypeantibody in combination with CPA pretreatment. The combination treatmentgroup demonstrated a statistically significant enhancement (p<0.050) ofanti-tumor activity when compared against the isotype and monotherapycontrol groups. Using an ELISPOT assay, the effects of treatment on themagnitude of CD8⁺ T cell responses directed against thewell-characterized p15E tumor antigen were measured. Both CPA andA09-246-2 showed substantially increased levels of p15E-reactive CD8⁺ Tcells (˜100 spots in both groups) when compared to the isotype control(˜25 spots), with the combination group showing a further enhancement(˜250 spots). Thus, the anti-tumor efficacy of the CPA plus A09-240-2combination was associated with increased frequencies of tumor-antigenreactive CTL.

4.5 Combination With Cyclophosphamide/Stimuvax

The ability of PD-L1 blockade to restore anti-tumor T cell responsesprovides a strong rationale for combination with cancer vaccines.Stimuvax is a vaccine against the human MUC1 antigen, which is commonlyoverexpressed by solid tumors. Mice transgenic for the human MUC1protein (MUC1.tg mice) are immunologically tolerant of the antigen, and,when inoculated with murine tumors that also express human MUC1, providea relevant model of the clinical vaccination setting. In the clinic,cyclophosphamide (CPA) pre-treatment is used in combination withStimuvax as a strategy for transiently depleting immunosuppressiveT_(reg) cells that can inhibit the vaccine response.

In this study, MUC1.tg mice were inoculated subcutaneously in the rightrear flank with 1×10⁶ MC38/MUC1 colon carcinoma cells. Five days aftertumor cell inoculation, mice were randomized into treatment groups(n=10) on day −3. On day −3, a 100 mg/kg dose a CPA was administered byi.v. administration. Vaccination was initiated on day 0 and was repeatedweekly. Anti-PD-L1 Ab (A09-246-2) was dosed by i.p. injection on days 0,3, and 6. Tumors were measured twice weekly. The combination ofCPA/Stimuvax and A09-246-2 demonstrated significantly enhanced (p<0.050)tumor growth inhibition when compared against treatment withCPA/Stimuvax. In a second study, 1×10⁶ PANC02/MUC1 cells were inoculatedinto the pancreas of MUC1.tg mice. Four days later, mice were randomizedinto groups (N=8) and treatment was initiated. The same treatmentschedule was applied as for the first study. The combination ofCPA/Stimuvax and anti-PD-L1 (A09-248-2) significantly increased meansurvival time (MST) when compared against treatment with CPA/Stimuvax(MST of 43.5 days vs. 70 days, P=0.0001). Immunophenotyping by FACSshowed a significant trend towards increased percentages of CD8⁺ T_(EM)and CD8⁺ T_(CM) in the combination group.

4.6 Combination With Fractionated Radiotherapy

Radiotherapy (RT) has been demonstrated to enhance the immunogenicity oftumor cells, through increased expression of MHC class I anddiversification of the intracellular peptide pool. To test anti-PD-L1antibody treatment in combination with radiotherapy, MC38 coloncarcinoma cells (1×10⁵) were inoculated intramuscularly into the rightquadriceps of C57BL/6 female mice. When tumors reached a mean volume of150 mm³, mice were sorted into treatment groups (N=8) on day 0. Thetumor-bearing legs were isolated and treated with 360 cGy of gammairradiation from a cesium-137 source on days 0, 1, 2, 3, and 4 (totaldose of 1800 cGy). Anti-PD-L1 Ab (A09-246-2) was dosed i.v. at 400 μg ondays 3, 6, and 9. The A09-246-2 and radiotherapy combination resulted ina high rate of tumor regressions, ultimately leading to 6/10 completeresponses (CR). Mice with CR were re-challenged by inoculation of MC38tumor cells, and 3/6 mice remained tumor-free seventy-four days afterthe re-challenge, indicating that effective immune memory was generatedby the combination therapy. Conversely, a control group treated with anisotype control antibody in combination with radiation showedsignificant tumor growth inhibition, but did not induce regressions.

A repeat of the RT and anti-PD-L1 (A09-246-2) combination study wasperformed, with the inclusion of a second combination therapy group inwhich the mice were systemically depleted of CD8⁺ T cells. Additionalimmunological readouts measured in this study included FACS-basedimmunophenotyping of splenocytes, in vivo proliferation analysis, andELISPOT assay. Again, the combination demonstrated synergistic efficacythat induced an initial phase of regression or stasis in all of thetumors. However, complete regression was only observed in ⅛ mice, withone other mouse experiencing a prolonged period of tumor stasis.Depletion of CD8⁺ T cells completely abrogated the synergy of thecombination, confirming that the mechanism involves the stimulation ofanti-tumor CD8⁺ T cell responses. This observation was further supportedby increased frequencies of CD8⁺ T cells reactive to the p15E tumorantigen. Immunophenotyping by FACS revealed increased percentages ofCD8⁺ T cell proliferation in spleens, and increased splenic percentagesof CD8⁺ T_(EM) and CD8+ T_(CM).

4.7 Combination With Core Components of the FOLFOX Regimen

FOLFOX is a combination chemotherapy regimen, consisting of felinioacid, 5-fluorouracil (5 FU), and oxaliplatin (OX), used in the treatmentof stage III coloretal cancer. The potential for combining anti-PD-L1with the core components of FOLFOX (5-fluorouracil and oxaliplatin) inthe subcutaneous MC38 colon carcinoma model were studied. Mice wereinoculated in the right subcutaneous flank with 1×10⁶ MC38 coloncarcinoma cells. When tumors reached a mean volume of ˜50 mm³ , micewere sorted into treatment groups (N=10) on day 0. 5-FU (60 mg/kg i.v.)and OX (5 mg/kg i.p.) were administered on days 0 and 14. Anti-PD-L1 Ab(A09-246-2) (400 μg i.v.) was given on days 3, 6, and 9. The combinationtreatment was observed to have significantly greater efficacy (p<0.050)when compared to A09-246-2 given alone, or 5-FU and OX given incombination with an isotype antibody. A repeat of the anti-PD-L1 Ab andFOLFOX combination study was performed and, again, the combinationdemonstrated significantly greater (p<0.050) anti-tumor activity thaneither of the monotherapy regimens.

FACS-based immunophenotyping conducted in these studies revealedincreases in several immunological markers consistent with a CD8⁺ T celldriven MOA, including increased splenic levels of p15E tumor antigenspecific CD8⁺ T cells, an increase in the splenic ratio of T_(EM) toregulatory T cells (T_(reg)), and increased splenic percentages ofCD8⁺PD-1⁺ T cells. Furthermore, the percentage of tumor infiltratingnatural killer (NK) cells and CD8⁺ T cells was observed to increasesignificantly in the combination group.

4.8 4-Week Repeat Dose Pilot Toxicity Study in Cynomolgus Monkey

Four groups of 2 male and 2 female cynomolgus monkeys were treated withanti human PD-L1 (A09-246-2) at dose levels of 0 (vehicle), 20, 60 and140 mg/kg by weekly intravenous infusion for total of 5 administrations.

The TK evaluation indicates that all animals were exposed to the testmaterial throughout the study. The exposure levels increased roughlyproportionally to dose increasing at both 1^(st) and 4^(th) dose,without any relevant accumulation or gender-dependency at any dose. Antidrug antibody were detected in 2/4 and ¼ monkeys at 20 and 140 mg/kglevels respectively. There was no premature animal death in the study.No treatment related changes were noted in the 20 and 60 mg/kg dosinggroups for all parameters evaluated in the study.

At the high dose level of 140 mg/kg, treatment related findings includeslight decrease of lymphocytes in haematology testing, slight decreasein lymphocyte count together with a decrease in NK cell count on studyday 30. There were no significant histological changes in majororgans/tissues except moderate perivascular hemorrhage andinflammation/vessel necrosis observed at local injection site at the 140mg/kg. There was no clear trend or change observed in multicytokineanalysis at this dose level. Based on the results from this study the NoObservable Adverse Effect Level (NOAEL) was identified as 140 mg/kg.

Conclusion: A09-246-2 was tolerated in cynomolgus monkey at dose levelsup to 140 mg/kg after receiving a total of 5 consecutive weekly doses.Injection site reactions with moderate severity ofsubcutaneous/perivascular and vascular inflammatory and degenerativechanges were observed at 140 mg/kg.

1-48. (canceled)
 49. An isolated anti-PD-L1 antibody or antigen bindingfragment thereof which binds to a functional epitope comprising residuesY56 and D61 of human PD-L1 (SEQ ID NO:28). 50-92. (canceled)
 93. Theisolated anti-PD-L1 antibody or antigen binding fragment of claim 49wherein the functional epitope further comprises residues E58, E60, Q66,R113 and M115 of human PD-L1 (SEQ ID NO:28).
 94. The isolated anti-PD-L1antibody or antigen binding fragment of claim 93, wherein the isolatedanti-PD-L1 antibody or antigen binding fragment further binds to aconformational epitope comprising residues 54-66 and 112-122 of humanPD-L1 (SEQ ID NO:28).
 95. The isolated anti-PD-L1 antibody or antigenbinding fragment of claim 49, wherein the constant region of theanti-PD-L1 antibody is IgG1.
 96. The isolated anti-PD-L1 antibody orantigen binding fragment of claim 49, 93, or 94, wherein the anti-PD-L1antibody comprises: a light chain variable region; and a heavy chainvariable region comprising HVR-H1, HVR-H2, and HVR-H3 sequences,wherein: (a) the HVR-H1 sequence is X₁YX₂MX₃ (SEQ ID NO:1); (b) theHVR-H2 sequence is SIYPSGGX₄TFYADX₅VKG (SEQ ID NO:2); and (c) the HVR-H3sequence is IKLGTVTTVX₆Y (SEQ ID NO:3); and further wherein: X₁ is K, R,T, Q, G, A, W, M, I or S; X₂ is V, R, K, L, M or I; X₃ is H, T, N, Q, A,V, Y, W, F or M; X₄ is F or I; X₅ is S or T; and X₆is E or D.
 97. Theisolated anti-PD-L1 antibody or antigen binding fragment of claim 96,wherein: (a) X₁ is M, I or S; X₂ is R, K, L, M or I; X₃ is F or M; X₄ isF or I; X₅ is S or T; and X₆ is E or D; (b) X₁ is M, I or S; X₂ is L, Mor I; X₃ is F or M; X₄ is I; X₅ is S or T; and X₆ is D; or (c) X₁ is S;X₂ is I; X₃ is M; X₄ is I; X₅ is T; and X₆ is D.
 98. The isolatedanti-PD-L1 antibody or antigen binding fragment of claim 96, wherein theheavy chain variable region comprises heavy chain framework sequencesHC-FR1, HC-FR2, HC-FR3 and HC-FR4 interposed between the HVRs, thusforming a sequence of the formula:(HC-FR1)-(HVR-H1)-(HC-FR2)-(HVR-H2)-(HC-FR3)-(HVR-H3)-(HC-FR4).
 99. Theisolated anti-PD-L1 antibody or antigen binding fragment of claim 98,wherein one or more of the heavy chain framework sequences is selectedfrom: (SEQ ID NO: 4) (a) HC-FR1 is EVQLLESGGGLVQPGGSLRLSCAASGFTFS;(SEQ ID NO: 5) (b) HC-FR2 is WVRQAPGKGLEWVS; (SEQ ID NO: 6)(c) HC-FR3 is RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAR;  or (SEQ ID NO: 7)(d) HC-FR4 is WGQGTLVTVSS.


100. The isolated anti-PD-L1 antibody or antigen binding fragment ofclaim 96, wherein the light chain variable region comprises HVR-L1,HVR-L2 and HVR-L3 sequences, wherein: (a) the HVR-L1 sequence isTGTX₇X₈DVGX₉YNYVS (SEQ ID NO: 8); (b) the HVR-L2 sequence isX₁₀VX₁₁X₁₂RPS (SEQ ID NO: 9); and (c) the HVR-L3 sequence isSSX₁₃TX₁₄X₁₅X₁₆X₁₇RV (SEQ ID NO: 10); and further wherein: X₇ is N or S;X₈ is T, R or S; X₉ is A or G; X₁₀ is E or D; X₁₁ is I, N or S; X₁₂ isD, H or N; X₁₃ is F or Y; X₁₄ is N or S; X₁₅ is R, T or S; X₁₆ is G orS; and X₁₇ is I or T.
 101. The isolated anti-PD-L1 antibody or antigenbinding fragment of claim 100, wherein: (a) X₇ is N or S; X₈ is T, R orS; X₉ is A or G; X₁₀ is E or D; X₁₁ is N or S; X₁₂ is N; X₁₃ is F or Y;X₁₄ is S; X₁₅ is S; X₁₆ is G or S; and X₁₇ is T; or (b) X₇ is S; X₈ isS; X₉ is G; X₁₀ is D; X₁₁ is S; X₁₂ is N; X₁₃ is Y; X₁₄ is S; X₁₅ is S;X₁₆ is S; and X₁₇ is T.
 102. The isolated anti-PD-L1 antibody or antigenbinding fragment of claim 100, wherein the light chain variable regioncomprises light chain framework sequences LC-FR1, LC-FR2, LC-FR3 andLC-FR4, interposed between the HVRs, thus forming a sequence of theformula: (LC-FR1)-(HVR-L1)-(LC-FR2)-(HVR-L2)-(LC-FR3)-(HVR-L3)-(LC-FR4).103. The isolated anti-PD-L1 antibody or antigen binding fragment ofclaim 102, wherein one or more of the light chain framework sequences isselected from: (SEQ ID NO: 11) (a) LC-FR1 is QSALTQPASVSGSPGQSITISC;(SEQ ID NO: 12) (b) LC-FR2 is WYQQHPGKAPKLMIY; (SEQ ID NO: 13)(c) LC-FR3 is GVSNRFSGSKSGNTASLTISGLQAEDEADYYC;  or (SEQ ID NO: 14)(d) LC-FR4 is FGTGTKVTVL.


104. The isolated anti-PD-L1 antibody or antigen binding fragment ofclaim 100, wherein the anti-PD-L1 antibody or antigen binding fragmentthereof further comprises a human or murine constant region.
 105. Theisolated anti-PD-L1 antibody or antigen binding fragment of claim 100,wherein the antibody binds to human, mouse, or cynomolgus monkey PD-L1,or wherein the antibody is capable of blocking the interaction betweenhuman, mouse, or cynomolgus monkey PD-L1 and the respective human,mouse, or cynomolgus monkey PD-1 receptors.
 106. The isolated anti-PD-L1antibody or antigen binding fragment of claim 96, wherein: (a) the heavychain variable region comprises the sequence: (SEQ ID NO: 24)EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYIMMWVRQAPGKGLEWVSSIYPSGGITFYADTVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARIK LGTVTTVDYWGQGTLVTVSS;

and (b) the light chain variable region comprises the sequence:(SEQ ID NO: 25) QSALTQPASVSGSPGQSITISCTGTSSDVGGYNYVSWYQQHPGKAPKLMIYDVSNRPSGVSNRFSGSKSGNTASLTISGLQAEDEADYYCSSYTSSSTRV FGTGTKVTVL.


107. The isolated anti-PD-L1 antibody or antigen binding fragment ofclaim 49, wherein the anti-PD-L1 antibody comprises: (a) a heavy chainvariable region (VH) comprising an HVR-H1, HVR-H2 and HVR-H3 of SEQ IDNO: 32; and (b) a light chain variable region (VL) comprising an HVR-L1,HVR-L2 and HVR-L3 of SEQ ID NO:
 33. 108. The isolated anti-PD-L1antibody or antigen binding fragment of claim 107, wherein the HVR-H1comprises the amino acid sequence of SEQ ID NO: 15, the HVR-H2 comprisesthe amino acid sequence of SEQ ID NO: 16, the HVR-H3 comprises the aminoacid sequence of SEQ ID NO: 17, the HVR-L1 comprises the amino acidsequence of SEQ ID NO: 18, the HVR-L2 comprises the amino acid sequenceof SEQ ID NO: 19, and the HVR-L3 comprises the amino acid sequence ofSEQ ID NO:
 20. 109. The isolated anti-PD-L1 antibody or antigen bindingfragment of claim 107, wherein the anti-PD-L1 antibody comprises: (a) aheavy chain comprising the amino acid sequence of SEQ ID NO: 32, or theamino acid sequence of SEQ ID NO: 32 without the C-terminal lysine; and(b) a light chain comprising the amino acid sequence of SEQ ID NO: 33.110. The isolated anti-PD-L1 antibody or antigen binding fragment ofclaim 107, wherein each HVR is defined in accordance with the Kabatdefinition, the Chothia definition, a combination of the Kabatdefinition and the Chothia definition, the AbM definition, or thecontact definition of HVR.